Compositions and methods comprising a ligand of chemerin R

ABSTRACT

The present invention relates to a G-protein coupled receptor and a novel ligand therefor. The invention provides screening assays for the identification of candidate compounds which modulate the activity of the G-protein coupled receptor, as well as assays useful for the diagnosis and treatment of a disease or disorder related to the dysregulation of G-protein coupled receptor signaling.

This application is a Divisional of U.S. Ser. No. 10/893,485, filed Jul.16, 2004, which is a continuation in part of U.S. Applicant Ser. No.10/603,566, filed Jun. 25, 2003, which is a continuation in part of U.S.application Ser. No. 10/201,187, filed on Jul. 23, 2002, which is acontinuation in part of International application PCT/EP02/07647, filedJul. 9, 2002, which claims priority to U.S. application Ser. No.09/905,253, filed Jul. 13, 2001, which claims priority under 35 U.S.C.§119(e) to U.S. Provisional application No. 60/303,858, filed Jul. 9,2001. The entire content of each of the foregoing is incorporated hereinin its entirety

FIELD OF THE INVENTION

The invention relates to the identification of the natural ligand forthe orphan G-Protein Coupled Receptor (GPCR) ChemerinR and uses thereofin diagnosis and immuno therapy of a disease.

BACKGROUND OF THE INVENTION

G-protein coupled receptors (GPCRs) are proteins responsible fortransducing a signal within a cell. GPCRs have usually seventransmembrane domains. Upon binding of a ligand to an extra-cellularportion or fragment of a GPCR, a signal is transduced within the cellthat results in a change in a biological or physiological property orbehaviour of the cell. GPCRs, along with G-proteins and effectors(intracellular enzymes and channels modulated by G-proteins), are thecomponents of a modular signaling system that connects the state ofintra-cellular second messengers to extra-cellular inputs.

GPCR genes and gene products can modulate various physiologicalprocesses and are potential causative agents of disease. The GPCRs seemto be of critical importance to both the central nervous system andperipheral physiological processes.

The GPCR protein superfamily is represented in five families: Family I,receptors typified by rhodopsin and the beta2-adrenergic receptor andcurrently represented by over 200 unique members; Family II, theparathyroid hormone/calcitonin/secretin receptor family; Family III, themetabotropic glutamate receptor family, Family IV, the CAMP receptorfamily, important in the chemotaxis and development of D. discoideum;and Family V, the fungal mating pheromone receptor such as STE2.

G proteins represent a family of heterotrimeric proteins composed of α,β and γ subunits, that bind guanine nucleotides. These proteins areusually linked to cell surface receptors (receptors containing seventransmembrane domains) for signal transduction. Indeed, following ligandbinding to the GPCR, a conformational change is transmitted to the Gprotein, which causes the α-subunit to exchange a bound GDP molecule fora GTP molecule and to dissociate from the βγ-subunits.

The GTP-bound form of the α, β and γ-subunits typically functions as aneffector-modulating moiety, leading to the production of secondmessengers, such as cAMP (e.g. by activation of adenyl cyclase),diacylglycerol or inositol phosphates.

Greater than 20 different types of α-subunits are known in humans. Thesesubunits associate with a small pool of β and γ subunits. Examples ofmammalian G proteins include Gi, Go, Gq, Gs and Gt. G proteins aredescribed extensively in Lodish et al., Molecular Cell Biology(Scientific American Books Inc., New York, N.Y., 1995; and also byDownes and Gautam, 1999, The G-Protein Subunit Gene Families. Genomics62:544-552), the contents of both of which are incorporated herein byreference.

Known and uncharacterized GPCRs currently constitute major targets fordrug action and development. There are ongoing efforts to identify new Gprotein coupled receptors which can be used to screen for new agonistsand antagonists having potential prophylactic and therapeuticproperties.

More than 300 GPCRs have been cloned to date, excluding the family ofolfactory receptors. Mechanistically, approximately 50-60% of allclinically relevant drugs act by modulating the functions of variousGPCRs (Cudermann et al., J. Mol. Med., 73:51-63, 1995).

ChemerinR, also called Dez [Sequence ID Nos: 1 (human polynucleotidesequence, FIG. 1); 2 (human amino acid sequence, FIG. 2); 3 (mousepolynucleotide sequence, FIG. 3); 4 (mouse amino acid sequence, FIG. 3);5 (rat polynucleotide sequence; FIG. 4); and 6 (rat amino acid sequence,FIG. 4)] has been described as an orphan G protein coupled receptorrelated to GPR-1 (38% overall amino acid identity), C3a receptor (38%),C5a anaphylatoxin receptor (36%) and formyl Met-Leu-Phe receptors (35%).ChemerinR is more distantly related to the chemokine receptors subfamily(Methner A, Hermey G, Schinke B, Hermans-Borgmeyer I. (1997) BiochemBiophys Res Commun 233:336-42; Samson M, Edinger A L, Stordeur P, RuckerJ, Verhasselt V, Sharron M, Govaerts C, Mollereau C, Vassart G, Doms RW, Parmentier M. (1998) Eur J Immunol 28:1689-700). ChemerinRtranscripts were found to be abundant in monocyte-derived dendriticcells and macrophages, with or without treatment with LPS. Lowexpression can also be detected by reverse transcription-PCR in CD4+ Tlymphocytes. In situ hybridization experiments also showed that thereceptor was differentially regulated during development, with aprominent expression in developing osseous and cartilaginous tissues. Itwas also detectable in the adult parathyroid glands, indicating apossible function in phosphocalic metabolism.

The gene encoding ChemerinR was assigned by radiation hybrid mapping tothe q21.2-21.3 region of human chromosome 12, outside the gene clustersidentified so far for chemoattractant receptors. ChemerinR was tested infusion assays for potential coreceptor activity by a range of HIV-1,HIV-2 and SIV viral strains. Several SIV strains (SIVmac316, SIVmac239,SIVmac17E-Fr and SIVsm62A), as well as a primary HIV-1 strain(92UG024-2) efficiently used ChemerinR as a co-receptor. This receptortherefore appears to be a coreceptor for immunodeficiency viruses thatdoes not belong to the chemokine receptor family. It is also a putativechemoattractant receptor and it could play an important role in therecruitment or trafficking of leukocyte cell populations. ChemerinR, byits specific expression in macrophages and immature dendritic cells,appears as a particularly attractive candidate receptor involved in theinitiation and early regulation of immune responses.

TIG2 (Tazarotene-induced gene 2, thereafter Preprochemerin [Sequence IDNos: 7 (human Preprochemerin polynucleotide sequence, FIG. 6); 8 (humanamino acid sequence, FIG. 6); 9 (mouse polynucleotide sequence, FIG. 7);and 10 (mouse amino acid sequence, FIG. 7)] was identified as a cDNA,the expression of which is up-regulated by the treatment of skin raftcultures by the retinoic acid receptor (RAR) beta/gamma-selectiveanti-psoriatic synthetic retinoid, tazarotene [AGN 190168/ethyl6-[2-(4,4-dimethylthiochroman-6-yl)-ethynyl]nicotinate] (Nagpal S, PatelS, Jacobe H, DiSepio D, Ghosn C, Malhotra M, Teng M, Duvic M,Chandraratna R A. (1997) J Invest Dermatol 109: 91-5). Theretinoid-mediated up-regulation in the expression of Preprochemerin wasconfirmed by Northern blot analysis. The Preprochemerin is located at17p13.3 position, a region associated with pancretic tumorigenesis. ThePreprochemerin cDNA is 830 bp long and encodes a putative proteinproduct of 163 amino acids. Preprochemerin is expressed and induced bytazarotene in culture only when keratinocytes and fibroblasts form atissue-like 3-dimensional structure. RAR-specific retinoids were alsoshown to increase Preprochemerin mRNA levels. In contrast, neitherRXR-specific retinoids nor 1,25-dihydroxyvitamin D3 increasedPreprochemerin levels in these cells. Preprochemerin is also expresssedat high levels in nonlesional psoriatic skin but at lower levels in thepsoriatic lesion and its expression is up-regulated in psoriatic lesionsafter topical application of tazarotene. In addition, Preprochemerin hasbeen shown to be dramatically upregulated by 1,25 dihydroxyvitamin D3and dexamethasone in osteoclast-supporting stromal cells (Adams A E,Abu-Amer Y, Chappel J, Stueckle S, Ross F P, Teitelbaum S L, Suva L J.(1999) J Cell Biochem 74: 587-95).

Dendritic cells (DCs) and macrophages are professionalantigen-presenting cells that play key roles in both innate and adaptiveimmunity. DCs and macrophages are attracted to infection andinflammatory sites by a variety of factors, among which chemokinesconstitute the largest group so far (Caux, C. et al. (2002)Transplantation 73: S7-S11, Mellman, I. and Steinman, R M (2001) Cell106:255-258). It has been shown that tremendous functional,morphological and metabolic diversity exists among these cellpopulations. One of these functional differences is the expression ofdifferential sets of chemoattractant receptors, which is responsible forthe selective recruitment of specific cell subpopulations, according totheir lineage, origin and maturation state (Caux, C. et al. (2002)Transplantation 73: S7-S11). Many tumor types have been demonstrated toattract macrophages and DCs through the direct or indirect production ofchemoattractant factors (Coussens, L M and Werb, Z. (2002) Nature420:860-867, Vicari, A P and Caux, C. (2002) Cytokine Growth FactorsRev. 13:143-154). These include a number of CC-chemokines, such asMCP-1.

DCs are specialized antigen-presenting cells located throughout thehuman body. DCs function as sentinels of the immune system. They serveas essential link between innate and adaptive immune systems and induceboth primary and secondary immune responses (Palucka, K A andBanchereau, J. (1999) J. Clin. Immunol. 19:12-25). They traffic from theblood to the tissues where, while immature, they capture antigens. Theythen leave the tissues and move to the draining lymphoid organs where,coverted into mature DCs, they initiate the immune response byactivating naïve CD8⁺ cells, which seek out and kill theantigen-expressing tumor cells. Chemokines are important effectors ofthe regulation of DCs recruitment, and depending on the chemokinegradient released at the site of injury, different DC populations willbe recruited. It is expected that the type of resulting immune responsewill likely be dependent on the DC subpopulation recruited and thus onthe chemokines secreted (Caux, C. et al. (2002) Transplantation 73:S7-S11).

SUMMARY OF THE INVENTION

The invention is based on the discovery that Chemerin, a polypeptideresulting from the proteolytic processing of the Proprechemerinprecursor, is a natural ligand of the ChemerinR, and binds specificallyto ChemerinR. The invention encompasses a class of polypeptide sequencesissued from the C-terminal end of Chemerin containing a sequence motifN1N2X1X2X3N3X4N4X5 wherein N1-N4 are aromatic or hydrophobic amino acidsand X1-X5 are any amino acid, as well as the nucleic acid sequencesencoding this sequence motif. In one embodiment, N1 and N2 are aromaticor hydrophobic amino acids, and N3 and N4 are hydrophobic amino acids.In one embodiment, the polypeptide comprises YFX1X2X3FX4FX5. In anotherembodiment, the polypeptide comprises YFPGQFAFS. In another embodiment,the polypeptide comprises QRAGEDPHSFYFPGQFAFS. In another embodiment,the polypeptide comprises an amino acid sequence selected from the groupconsisting of: LFPGQFAFS, IFPGQFAFS, FLPGQFAFS, YLPGQFAFS, YVPGQFAFS andYFPGQFAFD-CONH2.

The invention also encompasses the nucleic acid and polypeptidesequences of Chemerin from mammals. The invention further encompassesthe polynucleic acid and peptide sequences of truncated Chemerin. Theinvention further encompasses the functionally-equivalent analogs ofChemerin nucleic acid and polypeptide sequences that contain varioussubstitutions from the naturally-occurring sequences.

The invention further encompasses expressing vectors encodingpolypeptides that specifically bind to a ChemerinR polypeptide. In oneembodiment, the expressing vector encodes the polypeptide or peptidesequences comprising N1N2X1X2X3N3X4N4×5, wherein N1-N3 are aromatic orhydrophobic amino acids and X1-X5 are any amino acids. In anotherembodiment, the expressing vector encodes the polypeptide sequencescomprising YFX1X2X3FX4FX5. In another embodiment, the expressing vectorencodes the polypeptides comprising YFPGQFAFS. In another embodiment,the expressing vector encodes the polypeptides comprisingQRAGEDPHSFYFPGQFAFS (SEQ ID NO: 53). In another embodiment, theexpressing vector encodes a Preprochemerin polypeptide as depicted inSEQ ID NO: 47.

The invention further encompasses antibodies to a Chemerin polypeptide.In one embodiment, the antibody is polyclonal antibody. In anotherembodiment, the antibody is monoclonal antibody. In another embodiment,the monoclonal antibody specifically binds to an epitope comprisingFSKALPRS (SEQ ID NO 89).

The invention further encompasses a composition containing any one ofthe above identified polypeptides. The invention further encompasses acomposition containing any one of the above identified nucleic acidsequences. In one embodiment, the composition is a therapeuticcomposition containing the polypeptide/nucleic acid sequences in aacceptable carrier.

The invention further encompasses the use of the interaction ofChemerinR polypeptides and Chemerin polypeptides as the basis ofscreening assays for agents that modulate the activity of the ChemerinRreceptor.

The invention encompasses a method of identifying an agent thatmodulates the function of ChemerinR, the method comprising: a)contacting a ChemerinR polypeptide with a Chemerin polypeptide in thepresence and absence of a candidate modulator under conditionspermitting the binding of the Chemerin polypeptide to the ChemerinRpolypeptide; and b) measuring the binding of the ChemerinR polypeptideto the Chemerin polypeptide, wherein a decrease in binding in thepresence of the candidate modulator, relative to the binding in theabsence of the candidate modulator, identifies the candidate modulatoras an agent that modulates the function of ChemerinR.

The invention further encompasses a method of detecting the presence, ina sample, of an agent that modulates the function of ChemerinR in asample, the method comprising a) contacting a ChemerinR polypeptide witha Chemerin polypeptide in the presence and absence of the sample underconditions permitting the binding of the Chemerin polypeptide to theChemerinR polypeptide; and b) measuring the binding of the ChemerinRpolypeptide to the Chemerin polypeptide, wherein a decrease in bindingin the presence of the sample, relative to the binding in the absence ofthe candidate modulator, indicates the presence, in the sample of anagent that modulates the function of ChemerinR.

In a preferred embodiment of either of the preceding methods, themeasuring is performed using a method selected from label displacement,surface plasmon resonance, fluorescence resonance energy transfer,fluorescence quenching, and fluorescence polarization.

The invention further encompasses a method of identifying an agent thatmodulates the function of ChemerinR, the method comprising: a)contacting a ChemerinR polypeptide with a Chemerin polypeptide in thepresence and absence of a candidate modulator; and b) measuring asignaling activity of the ChemerinR polypeptide, wherein a change in theactivity in the presence of the candidate modulator relative to theactivity in the absence of the candidate modulator identifies thecandidate modulator as an agent that modulates the function ofChemerinR.

The invention further encompasses a method of identifying an agent thatmodulates the function of ChemerinR, the method comprising: a)contacting a ChemerinR polypeptide with a candidate modulator; b)measuring a signaling activity of the ChemerinR polypeptide in thepresence of the candidate modulator; and c) comparing the activitymeasured in the presence of the candidate modulator to the activitymeasured in a sample in which the ChemerinR polypeptide is contactedwith a Chemerin polypeptide at its EC₅₀, wherein the candidate modulatoris identified as an agent that modulates the function of ChemerinR whenthe amount of the activity measured in the presence of the candidatemodulator is at least 50% of the amount induced by the Chemerinpolypeptide present at its EC₅₀.

The invention further encompasses a method of detecting the presence, ina sample, of an agent that modulates the function of ChemerinR, themethod comprising: a) contacting a ChemerinR polypeptide with Chemerinpolypeptide in the presence and absence of the sample; b) measuring asignaling activity of the ChemerinR polypeptide; and c) comparing theamount of the activity measured in a reaction containing ChemerinR andChemerin polypeptides without the sample to the amount of the activitymeasured in a reaction containing ChemerinR, Chemerin and the sample,wherein a change in the activity in the presence of the sample relativeto the activity in the absence of the sample indicates the presence, inthe sample, of an agent that modulates the function of ChemerinR.

The invention further encompasses a method of detecting the presence, ina sample, of an agent that modulates the function of ChemerinR, themethod comprising: a) contacting a ChemerinR polypeptide with thesample; b) measuring a signaling activity of the ChemerinR polypeptidein the presence of the sample; and c) comparing the activity measured inthe presence of the sample to the activity measured in a reaction inwhich the ChemerinR polypeptide is contacted with a Chemerin polypeptidepresent at its EC₅₀, wherein an agent that modulates the function ofChemerinR is detected if the amount of the activity measured in thepresence of the sample is at least 50% of the amount induced by theChemerin polypeptide present at its EC₅₀.

In a preferred embodiment of each of the preceding methods, the Chemerinpolypeptide is detectably labeled. It is preferred that the Chemerinpolypeptide is detectably labeled with a moiety selected from the groupconsisting of a radioisotope, a fluorophore, a quencher of fluorescence,an enzyme, an affinity tag, and an epitope tag.

In one embodiment of any of the preceding methods, the contacting isperformed in or on a cell expressing the ChemerinR polypeptide.

In another embodiment of any of the preceding methods, the contacting isperformed in or on synthetic liposomes (see Tajib et al., 2000, NatureBiotechnology 18: 649-654, which is incorporated herein by reference) orvirus-induced budding membranes containing a ChemerinR polypeptide. (SeeWO0102551, 2001, incorporated herein by reference).

In another embodiment of any of the preceding methods, the method isperformed using a membrane fraction from cells expressing the ChemerinRpolypeptide.

In another embodiment, the agent is selected from the group consistingof a peptide, a polypeptide, an antibody or antigen-binding fragmentthereof, a lipid, a carbohydrate, a nucleic acid, and a small organicmolecule.

In another embodiment, the step of measuring a signaling activity of theChemerinR polypeptide comprises detecting a change in the level of asecond messenger.

In another embodiment, the step of measuring a signaling activitycomprises measurement of guanine nucleotide binding or exchange,adenylate cyclase activity, cAMP, Protein Kinase C activity,phosphatidylinosotol breakdown, diacylglycerol, inositol triphosphate,intracellular calcium, arachinoid acid, MAP kinase activity, tyrosinekinase activity, or reporter gene expression.

In a preferred embodiment, the measuring a signaling activity comprisesusing an aequorin-based assay.

The invention further encompasses a method of modulating the activity ofa ChemerinR polypeptide in a cell, the method comprising the step ofdelivering to the cell an agent that modulates the activity of aChemerinR polypeptide, such that the activity of ChemerinR is modulated.

The invention further encompasses a method of diagnosing a disease ordisorder characterized by dysregulation of ChemerinR signaling, themethod comprising: a) contacting a tissue sample with an antibodyspecific for a ChemerinR polypeptide; b) detecting binding of theantibody to the tissue sample; and c) comparing the binding detected instep (b) with a standard, wherein a difference in binding relative tothe standard is diagnostic of a disease or disorder characterized bydysregulation of ChemerinR.

The invention further encompasses a method of diagnosing a disease ordisorder characterized by dysregulation of ChemerinR signaling, themethod comprising: a) contacting a tissue sample with an antibodyspecific for a Chemerin polypeptide; b) detecting binding of theantibody to the tissue sample; and c) comparing the binding detected instep (b) with a standard, wherein a difference in binding relative tothe standard is diagnostic of a disease or disorder characterized bydysregulation of ChemerinR.

The invention also encompasses diagnostic assays based upon theChemerinR/Chemerin polypeptide interaction, as well as kits forperforming diagnostic and screening assays.

The invention further encompasses a method of diagnosing a disease ordisorder characterized by dysregulation of ChemerinR signaling, themethod comprising: a) contacting a tissue sample with an antibodyspecific for a ChemerinR polypeptide and an antibody specific for aChemerin polypeptide; b) detecting binding of the antibodies to thetissue sample; and c) comparing the binding detected in step (b) with astandard, wherein a difference in the binding of either antibody orboth, relative to the standard, is diagnostic of a disease or disordercharacterized by dysregulation of ChemerinR.

The invention further encompasses a method of diagnosing a disease ordisorder characterized by dysregulation of ChemerinR signaling, themethod comprising: a) isolating nucleic acid from a tissue sample; b)amplifying a ChemerinR polynucleotide, using the nucleic acid as atemplate; and c) comparing the amount of amplified ChemerinRpolynucleotide produced in step (b) with a standard, wherein adifference in the amount of amplified ChemerinR polynucleotide relativeto the standard is diagnostic of a disease or disorder characterized bydysregulation of ChemerinR. In a preferred embodiment, the step ofamplifying comprises RT/PCR. In another preferred embodiment, the stepof comparing the amount is performed on a microarray.

The invention further encompasses a method of diagnosing a disease ordisorder characterized by dysregulation of ChemerinR signaling, themethod comprising: a) isolating nucleic acid from a tissue sample; b)amplifying a ChemerinR polynucleotide, using the nucleic acid as atemplate; and c) comparing the sequence of the amplified ChemerinRpolynucleotide produced in step (b) with a standard, wherein adifference in the sequence, relative to the standard is diagnostic of adisease or disorder characterized by dysregulation of ChemerinR. In apreferred embodiment, the step of amplifying comprises RT/PCR. Inanother preferred embodiment, the standard is SEQ ID NO: 1. In anotherpreferred embodiment, the step of comparing the sequence comprisesminisequencing. In another preferred embodiment, the step of comparingthe sequence is performed on a microarray.

The invention further encompasses a method of diagnosing a disease ordisorder characterized by dysregulation of ChemerinR signaling, themethod comprising: a) isolating nucleic acid from a tissue sample; b)amplifying a Chemerin polynucleotide, using the nucleic acid as atemplate; and c) comparing the amount of amplified Chemerinpolynucleotide produced in step (b) with a standard, wherein adifference in the amount of amplified Chemerin polynucleotide relativeto the standard is diagnostic of a disease or disorder characterized bydysregulation of ChemerinR. In a preferred embodiment, the step ofamplifying comprises RT/PCR. In another preferred embodiment, the stepof comparing the amount is performed on a microarray.

The invention further encompasses a method of diagnosing a disease ordisorder characterized by dysregulation of ChemerinR signaling, themethod comprising: a) isolating nucleic acid from a tissue sample; b)amplifying a Chemerin polynucleotide, using the nucleic acid as atemplate; and c) comparing the sequence of the amplified Chemerinpolynucleotide produced in step (b) with a standard, wherein adifference in the sequence, relative to the standard is diagnostic of adisease or disorder characterized by dysregulation of ChemerinR. In apreferred embodiment, the step of amplifying comprises RT/PCR. Inanother preferred embodiment, the standard is SEQ ID NO: 7. In anotherpreferred embodiment, the step of comparing the sequence comprisesminisequencing. In another preferred embodiment, the step of comparingthe sequence is performed on a microarray.

The invention further encompasses a composition comprising an isolatedChemerinR polypeptide.

The invention further encompasses an antibody specific for a ChemerinRpolypeptide.

The invention further encompasses a kit for screening for agents thatmodulate ChemerinR signaling, or for the diagnosis of a disease ordisorder characterized by dysregulation of a ChemerinR polypeptide, thekit comprising an isolated ChemerinR polypeptide and packaging materialstherefor. In a preferred embodiment, the kit further comprises aChemerin polypeptide. Diagnostic kits according to the invention permitthe determination of whether, for example, a tissue sample or an extractprepared from a tissue sample has an elevated level or activity ofChemerin or ChemerinR. The kits also permit the identification ofmutations in genes encoding Chemerin or Chemerin and detection ofabnormal levels of nucleic acids encoding ChemerinR or Chemerin.

The invention further encompasses a kit for screening for agents thatmodulate ChemerinR signaling, or for the diagnosis of a disease ordisorder characterized by dysregulation of a ChemerinR polypeptide, thekit comprising an isolated polynucleotide encoding a ChemerinRpolypeptide and packaging materials therefor. In a preferred embodiment,the kit further comprises an isolated polynucleotide encoding a Chemerinpolypeptide.

The invention further encompasses a kit for screening for agents thatmodulate ChemerinR signaling, or for the diagnosis of a disease ordisorder characterized by dysregulation of a ChemerinR polypeptide, thekit comprising a cell transformed with a polynucleotide encoding aChemerinR polypeptide and packaging materials therefor. In a preferredembodiment, the kit further comprises an isolated polynucleotideencoding a Chemerin polypeptide or a cell comprising a polynucleotideencoding a Chemerin polypeptide.

The invention further encompasses a non-human mammal having a homozygousnull mutation in the gene encoding ChemerinR.

The invention further encompasses a non-human mammal transgenic for aChemerinR polynucleotide.

The invention further encompasses a non-human mammal transgenic for aChemerin polynucleotide.

The invention further encompasses a method for gene transfer ofPreprochemerin (SEQ ID NO: 7) or a gene transfer of truncatedPreprochemerin (SEQ ID NO: 72) into a cell. The invention furtherencompasses a method for gene transfer of Preprochemerin or a genetransfer of truncated Preprochemerin directly into tissues in vivo fortreatment of a disease or disorder. The gene transfer may employ DNAexpressing plasmid vectors, or viral vectors, or non-viral gene transfertools such as liposomes, receptor-mediated endocytosis, and gene gun. Inone particular embodiment, the vector is expressed in a tissue-specificand tumor-selective manner.

The invention further encompasses an ex vivo gene therapy with the geneencoding the Preprochemerin or the gene encoding truncatedPreprochemerin.

The invention further encompasses an ex vivo gene transfection ofPreprochemerin or truncated Preprochemerin into a disease cell and thesubsequent graft of the transfected cell in vivo for assaying theanti-disease effect of Preprochemerin or truncated Preprochemerin invivo.

The invention further encompasses an in vivo gene therapy with the geneencoding the Preprochemerin or truncated Preprochemerin. One embodimentof the invention includes administering the gene encoding Preprochemerinor truncated Preprochemerin polynucleotides into a subject forstimulating immuno response or therapeutic treatment of a disease.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the nucleotide (SEQ ID NO: 1) and deduced amino acidsequence of human ChemerinR/Dezb/CMKRL1 according to one embodiment ofthe invention.

FIG. 2 shows the amino acid sequence of human ChemerinR/Dezb/CMKRL1 (SEQID NO: 2) according to one embodiment of the invention. The sevenpredicted transmembrane domains are underlined. The consensus sequencefor N-linked glycosylation (N—X—S/T) in the N terminus is bold, and thepotential site of phosphorylation by PKC (S/T-X—R/K) in the C terminusis italicized.

FIG. 3 shows the nucleotide (SEQ ID NO:3) and deduced amino acid (SEQ IDNO: 4) sequences of mouse Dez, the mouse orthologue of ChemerinRaccording to one embodiment of the invention.

FIG. 4 shows that nucleotide (SEQ ID NO: 5) and deduced amino acid (SEQID NO: 6) sequences of rat G-Protein-Coupled Chemoattractant-1, the ratorthologue of ChemerinR/Dezb/CMKRL1 according to one embodiment of theinvention.

FIG. 5 shows the structural similarities between the amino acidsequences of ChemerinR/Dezb/CMKRL1 and the sequences of AT2, C3a, c5a,and fMLP receptors and selected chemokine receptor sequences performedusing the ClustalX algorithm according to one embodiment of theinvention. The dendrogram shown was constructed using the TreeViewAlgorithm.

FIG. 6 shows the nucleotide (SEQ ID NO: 7) and deduced amino acid (SEQID NO: 8) sequences of human Preprochemerin according to one embodimentof the invention.

FIG. 7 shows the nucleotide (SEQ ID NO: 9) and deduced amino acid (SEQID NO: 10) sequences of mouse Preprochemerin according to one embodimentof the invention.

FIG. 8 shows the nucleotide (SEQ ID NO: 11) and deduced amino acid (SEQID NO: 12) sequences of human Prochemerin according to one embodiment ofthe invention.

FIG. 9 shows the nucleotide (SEQ ID NO: 13) and deduced amino acid (SEQID NO: 14) sequences of human Chemerin according to one embodiment ofthe invention.

FIG. 10 shows an alignment of the human and mouse Preprochemerin aminoacid sequences according to one embodiment of the invention. Identicalamino acids are conservative substitutions are boxed.

FIG. 11 shows an alignment of human, mouse, rat, sus, bos and GallusPreprochemerin sequences according to one embodiment of the invention.The figure provides the percent amino acide identity across any twospecies listed.

FIG. 12 shows a partial chromatogram of the fifth step of purificationof Chemerin from ascitic fluid according to one embodiment of theinvention. The active fractions (eluted with approximately 28% CH₃CN) ofthe previous step were diluted 6 fold with 0.1% TFA in H₂O and directlyloaded onto a C18 reverse phase column (1 mm×50 mm, Vydac)pre-equilabrated with 5% CH₃CN/0.1% TFA in H₂O at a flow-rate of 0.1ml/min. at room temperature. A 5-95% gradient of CH₃CN in 0.1% TFA wasapplied with a 0.3%/min slope between 25 and 45%. The activity waseluted at 40% CH₃CN (indicated by the black horizontal line).

FIG. 13 shows the identification of a specific response for ChemerinRfollowing screening of HPLC fractions obtained from the fractionation ofhuman ovary ascites according to one embodiment of the invention. Thedifferent fractions obtained following fractionation of human ovaryascites were diluted fivefold in the assay buffer and tested in anaequorin assay using a cell line expressing ChemerinR (open circles) orcell lines expressing unrelated receptors (closed triangles andsquares). The response obtained for each fraction was normalized usingthe ATP response of each cell line.

FIG. 14 shows the activation of ChemerinR by conditioned medium of 293Tcells transiently transfected with Chemerin according to one embodimentof the invention. 293T cells were transiently transfected withpCDNA3-Preprochemerin (TIG 2) or with pCDNA3 alone (mock transfected).Increasing volumes of the supernatant collected 4 days aftertransfection were analyzed using a Microlumat in an aequorin-based assaywith CHO cells expressing ChemerinR. The assay was performed intriplicate, and SD is indicated. A representative experiment is shown.

FIG. 15 shows the characterization of antibodies directed againstChemerinR by flow cytometry according to one embodiment of theinvention.

FIG. 16 shows the polypeptide (SEQ ID NO: 73) and polynucleotide (SEQ IDNO: 72) of the truncated human Preprochemerin according to oneembodiment of the invention.

FIG. 17 shows the EC₅₀ for activation of ChemerinR by truncated humanPreprochemerin (truncated hTIG2) according to one embodiment of theinvention.

FIG. 18 shows the tissue distribution of hPreprochemerin mRNA accordingto one embodiment of the invention.

FIG. 19 shows the tissue distribution of ChemerinR mRNA according to oneembodiment of the invention.

FIG. 20 a shows the human polypeptides C-terminal extented or truncatedfrom human chemerin-19 peptide according to one embodiment of theinvention.

FIG. 20 b shows the mouse Chemerin polypeptides according to oneembodiment of the invention.

FIG. 21 shows the isolation of human Chemerin from human inflammatoryfluid according to one embodiment of the invention.

FIGS. 22A and B show fractions and sequences of major peaks from massspectrometer spectrum according to one embodiment of the invention. FIG.20C shows Chemerin polypeptide sequence alignment.

FIG. 23A shows SDS/PAGE of human recombinant Chemerin, expressed inCHO-K1 cells and purified by HPLC according to one embodiment of theinvention. The gel was silver stained and the major band corresponds toa protein of 18 kDa.Mass spectrometry analysis demonstrated the cleavageof the six C-terminal amino acids in this biologically active protein.FIGS. 23B-F show the functional assays of human recombinant Chemerin.

FIGS. 24A-F show expression and tissue distribution of human Chemerinand its receptor according to one embodiment of the invention.

FIG. 25A-D show the biological activity of truncated Chemerin peptidesas in aequorin assay according to one embodiment of the invention.

FIGS. 26A-H show the biological activity of Chemerin ex vivo on primarycells according to one embodiment of the invention.

FIG. 27 shows the anti-tumor activity of mouse Chemerin in vivoaccording to one embodiment of the invention.

FIG. 28 shows the biological activity of LFPGQFAFS on Chemerin Raccording to one embodiment of the invention.

FIG. 29 shows the biological activity of IFPGQFAFS on Chemerin Raccording to one embodiment of the invention.

FIG. 30 shows the biological activity of FLPGQFAFS on Chemerin Raccording to one embodiment of the invention.

FIG. 31 shows the biological activity of YLPGQFAFS on Chemerin Raccording to one embodiment of the invention.

FIG. 32 shows the biological activity of YVPGQFAFS on Chemerin Raccording to one embodiment of the invention.

FIG. 33 shows the biological activity of YFPGQFAFD-CONH2 on Chemerin Raccording to one embodiment of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to the discovery that Chemerin polypeptide is anatural ligand for ChemerinR and that the interaction between Chemerinand ChemerinR induces anti-disease immuno-responses. The interaction isuseful for screening assays for agents that modulate the interaction andthus the function of ChemerinR. The interaction between Chemerin andChemerinR also provides for the diagnosis of conditions involvingdysregulated receptor activity. The interaction also provides fortherapeutic approaches for treatment of a disease or disorder.

DEFINITIONS

For convenience, the meaning of certain terms and phrases used in thespecification, examples, and appended claims, are provided below.

The term “polypeptide” refers to a polymer in which the monomers areamino acids and are joined together through peptide or disulfide bonds.It also refers to either a full-length naturally-occurring amino acidsequence or a fragment thereof between about 8 and about 500 amino acidsin length. Additionally, unnatural amino acids, for example, β-alanine,phenyl glycine and homoarginine may be included. Commonly-encounteredamino acids which are not gene-encoded may also be used in the presentinvention. All of the amino acids used in the present invention may beeither the D- or L-optical isomer. The L-isomers are preferred.

As used herein, the term “ChemerinR polypeptide” refers to a polypeptidehaving two essential properties: 1) a ChemerinR polypeptide has at least70% amino acid identity, and preferably 80%, 90%, 95% or higher,including 100% amino acid identity, to SEQ ID NO: 2; and 2) a ChemerinRpolypeptide has ChemerinR activity, i.e., the polypeptide binds aChemerin polypeptide or a functional fragment thereof. Optimally, a“ChemerinR polypeptide” also has ChemerinR signaling activity as definedherein.

The term “a Chemerin polypeptide” refers to a polypeptide having atleast 30% or higher identity to a polypeptide selected from the groupconsisting of: SEQ ID NO: 14, SEQ ID NO: 73, SEQ ID NO: 61 and SEQ IDNos. 92-97, and the defined polypeptide specifically binds to andactivates a signaling activity of a ChemerinR polypeptide. Preferrably,the polypeptide is at least 50%, or higher identity to a polypeptideselected from the group consisting of: SEQ ID NO: 14, SEQ ID NO: 73, SEQID NO: 61 and SEQ ID Nos. 92-97. Preferrably, the polypeptide is atleast 60%, or 70%, or 80%, or 85%, or higher identity to a polypeptideselected from the group consisting of: SEQ ID NO: 14, SEQ ID NO: 73, SEQID NO: 61 and SEQ ID Nos. 92-97. The term “specifically binds” meansthat the Chemerin polypeptide has an EC₅₀, IC₅₀, or a K_(d), of 100 nMor less. “Chemerin polypeptide” also refers to a fragment of apolypeptide meeting the preceding definition, wherein the fragmentretains at least 50% of the binding activity and level of signalingactivation of the full length polypeptide of SEQ ID NO: 14. A Chemerinalso includes a anolog, variant or some short polypeptide fromC-terminal end of the Chemerin (SEQ ID NO 14) as depicted in FIGS. 8,and 16, 20 a and 20 b that binds specifically to a ChemerinRpolypeptide. A Chemerin polypeptide can comprise additions, insertions,deletions or substitutions relative to SEQ ID NO: 14, as long as theresulting polypeptide retains at least 50% of the binding activity andlevel of signaling activation of the full length polypeptide representedby SEQ ID NO: 14. In one embodiment, a “Chemerin polypeptide”encompasses further the truncated Preprochemerin sequence of SEQ ID NO:73 shown in FIG. 16 (the nucleotide sequence shown in FIG. 16, whichencodes the truncated Preprochemerin polypeptide is SEQ ID NO: 72). Inaddition to the sequences necessary for binding to ChemerinR andactivating a ChemerinR signaling actitity, a Chemerin polypeptide,including the truncated Chemerin polypeptide can comprise additionalsequences, as in for example, a Chemerin fusion protein. Non-limitingexamples of fusion partners include glutathione-S-transferase (GST),maltose binding protein, alkaline phosphatase, thioredoxin, greenfluorescent protein (GFP), histidine tags (e.g., 6× or greater H is), orepitope tags (e.g., Myc tag, FLAG tag).

The term “a nucleic acid sequence” refers to a polynucleotides such DNAor RNA. The term should also include both single and doublestrandedpolynucleotides. The term should also be understood to include, asequivalents, analogs of either RNA or DNA made from nucleotide analogs,and, as applicable to the embodiment being described, single (sense orantisense) and double-stranded polynucleotides. ESTs, chromosomes,cDNAs, mRNAs, and rRNAs are representative examples of molecules thatmay be referred to as nucleic acids.

As used herein, the term “Chemerin polynucleotide” refers to apolynucleotide that encodes a Chemerin polypeptide as defined herein, orthe complement thereof. In one embodiment, a “Chemerin polynucleotide”is a polynucleotide sequence which encodes a truncated Preprochemerinpolypeptide (e.g., the truncated Preprochemerin polypeptide shown inFIG. 17), such as the polynucleotide sequence shown in FIG. 17 (SEQ IDNO: 49).

As used herein, the term “a ChemerinR polynucleotide” refers to apolynucleotide that encodes a ChemerinR polypeptide, or a ChemerinRpolypeptide analog or variant as defined herein.

As used herein, the term “standard” refers to a sample taken from anindividual who is not affected by a disease or disorder characterized bydysregulation of G-protein coupled receptor (i.e., ChemerinR) activity.The “standard” is used as a reference for the comparison of receptormRNA or polypeptide levels and quality (i.e., mutant vs. wild type), aswell as for the comparison of G-protein coupled receptor activities. A“standard” also encompasses a reference sequence, e.g., SEQ ID NO: 1,with which sequences of nucleic acids or their encoded polypeptides arecompared.

As used herein, the term “dysregulation” refers to the signalingactivity of ChemerinR in a sample wherein a) a 10% or greater increaseor decrease in the amount of one or more of ChemerinR polypeptide,ligand or mRNA level is measured relative to a standard, as definedherein, in a given assay or; b) at least a single base pair change inthe ChemerinR coding sequence is detected relative to SEQ ID NO: 1, andresults in an alteration of ChemerinR ligand binding or signalingactivity as defined in paragraphs a), c) or d) or; c) a 10% or greaterincrease or decrease in the amount of ChemerinR ligand binding activityis measured relative to a standard, as defined herein, in a given assayor; d) a 10% or greater increase or decrease in a second messenger, asdefined herein, is measured relative to the standard, as defined herein,in a given assay.

The term “expression vector” refers to a nucleic acid construct capableof directing the expression of genes to which they are linked. Theconstruct further includes regulatory sequences, including for example,a promoter, operably linked to the genes. In general, expressing vectorsof utility in recombinant DNA techniques are often in the form of“plasmids” which refer generally to circular double stranded DNA loopswhich, in their vector form are not bound to chromosome.

The term “plasmid DNA expression vector” refers generally to a circulardouble stranded DNA loop which in their vector form are not bound to thechromosome, and which are capable of autonomous replication and/orexpression of nucleic acids to which it is linked.

The term “adenovirus expression vector” refers to an expression vectorwhich is derived from human adenovirus serotype 5, lacks ability toself-replicate, is capable of delivering into a cell a gene, and iscapable of autonomous replication and/or expression of the gene insidethe cell.

The term “composition” refers to a compound that is made of one or moremolecules, preferably a protein or a nucleic acid encoding a protein, ora mixture thereof. A composition can be naturally occurring, or derivedby recombinant technology, or by other synthetic means known to oneskill in the art.

The term “therapeutic composition” refers to a composition that upondelivered into a cell, acts upon the cell to correct or compensate foran underlying molecular deficit, or counteract a disease state orsyndrome of the cell.

The term “antibody” refers to the conventional immunoglobulin molecule,as well as fragments thereof which are also specifically reactive withone of the subject polypeptides. Antibodies can be fragmented usingconventional techniques and the fragments screened for utility in thesame manner as described herein below for whole antibodies. For example,F(ab)₂ fragments can be generated by treating antibody with pepsin. Theresulting F(ab)₂ fragment can be treated to reduce disulfide bridges toproduce Fab fragments. The antibody of the present invention is furtherintended to include bispecific, single-chain, and chimeric and humanizedmolecules having affinity for a polypeptide conferred by at least oneCDR region of the antibody. In preferred embodiments, the antibodies,the antibody further comprises a label attached thereto and able to bedetected, (e.g., the label can be a radioisotope, fluorescent compound,chemiluminescent compound, enzyme, or enzyme co-factor).

The term “monoclonal antibody” refers to an antibody that recognizesonly one type of antigen. This type of antibodies is produced by thedaughter cells of a single antibody-producing hybridoma.

The term “transgenic animal” refers to any animal, preferably anon-human mammal, bird, fish or an amphibian, in which one or more ofthe cells of the animal contain heterologous nucleic acid introduced byway of human intervention, such as by transgenic techniques well knownin the art. The nucleic acid is introduced into the cell, directly orindirectly by introduction into a precursor of the cell, by way ofdeliberate genetic manipulation, such as by microinjection or byinfection with a recombinant virus. The term genetic manipulation doesnot include classical cross-breeding, or in vitro fertilization, butrather is directed to the introduction of a recombinant DNA molecule.This molecule may be integrated within a chromosome, or it may beextra-chromosomally replicating DNA. In the typical transgenic animalsdescribed herein, the transgene causes cells to express a recombinantform of one of the subject polypeptide, e.g. either agonistic orantagonistic forms. However, transgenic animals in which the recombinantgene is silent are also contemplated, as for example, the FLP or CRErecombinase dependent constructs described below. Moreover, “transgenicanimal” also includes those recombinant animals in which gene disruptionof one or more genes is caused by human intervention, including bothrecombination and antisense techniques.

The term “therapeutically effective amount” refers to the total amountof each active component of the pharmaceutical composition or methodthat is sufficient to show a meaningful patient benefit, i.e.,treatment, healing, prevention or amelioration of the relevant medicalcondition, or an increase in rate of treatment, healing, prevention oramelioration of such conditions. When applied to an individual activeingredient, administered alone, the term refers to that ingredientalone. When applied to a combination, the term refers to combinedamounts of the active ingredients that results in the therapeuticeffect, whether administered in combination, serially or simultaneously.Generally, a composition will be administered in a single dosage in therange of 100 μg-100 mg/kg body weight, preferably in the range of 1μg-100 μg/kg body weight. This dosage may be repeated daily, weekly,monthly, yearly, or as considered appropriate by the treating physician.

As used herein, the term “ChemerinR activity” refers to specific bindingof a Chemerin polypeptide or a functional fragment thereof by aChemerinR polypeptide.

As used herein, the term “ChemerinR signaling activity” refers to theinitiation or propagation of signaling by a ChemerinR polypeptide.ChemerinR signaling activity is monitored by measuring a detectable stepin a signaling cascade by assaying one or more of the following:stimulation of GDP for GTP exchange on a G protein; alteration ofadenylate cyclase activity; protein kinase C modulation;phosphatidylinositol breakdown (generating second messengersdiacylglycerol, and inositol triphosphate); intracellular calcium flux;activation of MAP kinases; modulation of tyrosine kinases; or modulationof gene or reporter gene activity. A detectable step in a signalingcascade is considered initiated or mediated if the measurable activityis altered by 10% or more above or below a baseline established in thesubstantial absence of a Chemerin polypeptide relative to any of theChemerinR activity assays described herein below. The measurableactivity can be measured directly, as in, for example, measurement ofcAMP or diacylglycerol levels. Alternatively, the measurable activitycan be measured indirectly, as in, for example, a reporter gene assay.

As used herein, the term “detectable step” refers to a step that can bemeasured, either directly, e.g., by measurement of a second messenger ordetection of a modified (e.g., phosphorylated) protein, or indirectly,e.g., by monitoring a downstream effect of that step. For example,adenylate cyclase activation results in the generation of cAMP. Theactivity of adenylate cyclase can be measured directly, e.g., by anassay that monitors the production of cAMP in the assay, or indirectly,by measurement of actual levels of cAMP.

As used herein, the term “isolated” refers to a population of molecules,e.g., polypeptides or polynucleotides, the composition of which is lessthan 50% (by weight), preferably less than 40% and most preferably 2% orless, contaminating molecules of an unlike nature. When the term“isolated” is applied to a ChemerinR polypeptide, it is specificallymeant to encompass a ChemerinR polypeptide that is associated with orembedded in a lipid membrane.

As used herein, the terms “candidate compound” and “candidate modulator”refer to a composition being evaluated for the ability to modulateligand binding to a ChemerinR polypeptide or the ability to modulate anactivity of a ChemerinR polypeptide. Candidate modulators can be naturalor synthetic compounds, including, for example, small molecules,compounds contained in extracts of animal, plant, bacterial or fungalcells, as well as conditioned medium from such cells.

As used herein, the term “small molecule” refers to a compound havingmolecular mass of less than 3000 Daltons, preferably less than 2000 or1500, still more preferably less than 1000, and most preferably lessthan 600 Daltons. A “small organic molecule” is a small molecule thatcomprises carbon.

As used herein, the term “change in binding” or “change in activity” andthe equivalent terms “difference in binding” or “difference in activity”refer to an at least 10% increase or decrease in binding, or signalingactivity in a given assay.

As used herein, the term “conditions permitting the binding of Chemerinto ChemerinR” refers to conditions of, for example, temperature, saltconcentration, pH and protein concentration under which Chemerin bindsChemerinR. Exact binding conditions will vary depending upon the natureof the assay, for example, whether the assay uses viable cells or onlymembrane fraction of cells. However, because ChemerinR is a cell surfaceprotein, and because Chemerin is a secreted polypeptide that interactswith ChemerinR on the cell surface, favored conditions will generallyinclude physiological salt (90 mM) and pH (about 7.0 to 8.0).Temperatures for binding can vary from 15° C. to 37° C., but willpreferably be between room temperature and about 30° C. Theconcentration of Chemerin and ChemerinR polypeptide in a bindingreaction will also vary, but will preferably be about 0.1 pM (e.g., in areaction with radiolabeled tracer Chemerin, where the concentration isgenerally below the K_(d)) to 1 μM (e.g., Chemerin as competitor). As anexample, for a binding assay using ChemerinR-expressing cells andpurified, recombinant, labeled Chemerin polypeptide, binding isperformed using 0.1 nM labeled Chemerin, 100 nM cold Chemerin, and25,000 cells at 27° C. in 250 μl of a binding buffer consisting of 50 mMHEPES (pH 7.4), 1 mM CaCl₂, and 0.5% Fatty acid free BSA.

As used herein, the term “sample” refers to the source of moleculesbeing tested for the presence of an agent that modulates binding to orsignaling activity of a ChemerinR polypeptide. A sample can be anenvironmental sample, a natural extract of animal, plant yeast orbacterial cells or tissues, a clinical sample, a synthetic sample, or aconditioned medium from recombinant cells or a fermentation process. Theterm “tissue sample” refers to a tissue that is tested for the presence,abundance, quality or an activity of a ChemerinR polypeptide, a Chemerinpolypeptide, a nucleic acid encoding a ChemerinR or Chemerinpolypeptide, or an agent that modifies the ligand binding or activity ofa ChemerinR polypeptide.

As used herein, a “tissue” is an aggregate of cells that perform aparticular function in an organism. The term “tissue” as used hereinrefers to cellular material from a particular physiological region. Thecells in a particular tissue can comprise several different cell types.A non-limiting example of this would be brain tissue that furthercomprises neurons and glial cells, as well as capillary endothelialcells and blood cells, all contained in a given tissue section orsample. In addition to solid tissues, the term “tissue” is also intendedto encompass non-solid tissues, such as blood.

As used herein, the term “membrane fraction” refers to a preparation ofcellular lipid membranes comprising a ChemerinR polypeptide. As the termis used herein, a “membrane fraction” is distinct from a cellularhomogenate, in that at least a portion (i.e., at least 10%, andpreferably more) of non-membrane-associated cellular constituents hasbeen removed. The term “membrane associated” refers to those cellularconstituents that are either integrated into a lipid membrane or arephysically associated with a component that is integrated into a lipidmembrane.

As used herein, the term “decrease in binding” refers to a decrease ofat least 10% in the binding of a Chemerin polypeptide or other agonistto a ChemerinR polypeptide as measured in a binding assay as describedherein.

As used herein, the term “second messenger” refers to a molecule,generated or caused to vary in concentration by the activation of aG-Protein Coupled Receptor, that participates in the transduction of asignal from that GPCR. Non-limiting examples of second messengersinclude cAMP, diacylglycerol, inositol triphosphates and intracellularcalcium. The term “change in the level of a second messenger” refers toan increase or decrease of at least 10% in the detected level of a givensecond messenger relative to the amount detected in an assay performedin the absence of a candidate modulator.

As used herein, the term “aequorin-based assay” refers to an assay forGPCr activity that measures intracellular calcium flux induced byactivated GPCRs, wherein intracellular calcium flux is measured by theluminescence of aequorin expressed in the cell.

As used herein, the term “binding” refers to the physical association ofa ligand (e.g., a Chemerin polypeptide) with a receptor (e.g.,ChemerinR). As the term is used herein, binding is “specific” if itoccurs with an EC₅₀ or a K_(d) of 100 nM or less, generally in the rangeof 100 nM to 10 pM. For example, binding is specific if the EC₅₀ orK_(d) is 100 nM, 50 nM, 10 nM, 1 nM, 950 pM, 900 pM, 850 pM, 800 pM, 750pM, 700 pM, 650 pM, 600 pM, 550 pM, 500 pM, 450 pM, 400 pM, 350 pM, 300pM, 250 pM, 200 pM, 150 pM, 100 pM, 75 pM, 50 pM, 25 pM or 10 pM orless.

As used herein, the term “EC₅₀,” refers to that concentration of anagent at which a given activity, including binding of a Chemerinpolypeptide or other ligand and a functional activity of a ChemerinRpolypeptide, is 50% of the maximum for that ChemerinR activitymeasurable using the same assay. Stated differently, the “EC₅₀” is theconcentration of agent that gives 50% activation, when 100% activationis set at the amount of activity that does not increase with theaddition of more agonist. It should be noted that the “EC₅₀ of aChemerin polypeptide” will vary with the identity of the Chemerinpolypeptide; for example, variant Chemerin polypeptides (i.e., thosecontaining insertions, deletions, substitutions or fusions with otherpolypeptides, including Chemerin molecules from species other thanhumans and variants of them that satisfy the definition of Chemerinpolypeptide set forth above) can have EC₅₀ values higher than, lowerthan or the same as wild-type Chemerin. Therefore, where a Chemerinvariant sequence differs from wild-type Chemerin of SEQ ID NO:8, one ofthe skill in the art can determine the EC₅₀ for that variant accordingto conventional methods. The EC₅₀ of a given Chemerin polypeptide ismeasured by performing an assay for an activity of a fixed amount ofChemerinR polypeptide in the presence of doses of the Chemerinpolypeptide that increase at least until the ChemerinR response issaturated or maximal, and then plotting the measured ChemerinR activityversus the concentration of Chemerin polypeptide.

As used herein, the term “IC₅₀” is the concentration of an antagonist orinverse agonist that reduces the maximal activation of a ChemerinRreceptor by 50%.

As used herein, the term “detectably labeled” refers to the property ofa molecule, e.g., a Chemerin polypeptide or other ChemerinR ligand, thathas a structural modification that incorporates a functional group(label) that can be readily detected. Detectable labels include but arenot limited to fluorescent compounds, isotopic compounds,chemiluminescent compounds, quantum dot labels, biotin, enzymes,electron-dense reagents, and haptens or proteins for which antisera ormonoclonal antibodies are available. The various means of detectioninclude but are not limited to spectroscopic, photochemical,radiochemical, biochemical, immunochemical, or chemical means.

As used herein, the term “affinity tag” refers to a label, attached to amolecule of interest (e.g., a Chemerin polypeptide or other ChemerinRligand), that confers upon the labeled molecule the ability to bespecifically bound by a reagent that binds the label. Affinity tagsinclude, but are not limited to an epitope for an antibody (known as“epitope tags”), biotin, 6×His, and GST. Affinity tags can be used forthe detection, as well as for the purification of the labeled species.

As used herein, the term “decrease in binding” refers to a decrease ofat least 10% in the amount of binding detected in a given assay with aknown or suspected modulator of ChemerinR relative to binding detectedin an assay lacking that known or suspected modulator.

As used herein, the term “delivering,” when used in reference to a drugor agent, means the addition of the drug or agent to an assay mixture,or to a cell in culture. The term also refers to the administration ofthe drug or agent to an animal. Such administration can be, for example,by injection (in a suitable carrier, e.g., sterile saline or water) orby inhalation, or by an oral, transdermal, rectal, vaginal, or othercommon route of drug administration.

As used herein, the term “effective amount” refers to that amount of adrug or ChemerinR modulating agent that results in a change in aChemerinR activity as defined herein (i.e., at least 10% increase ordecrease in a ChemerinR activity).

As used herein, the term “amplifying,” when applied to a nucleic acidsequence, refers to a process whereby one or more copies of a nucleicacid sequence is generated from a template nucleic acid. A preferredmethod of “amplifying” is PCR or RT/PCR.

As used herein, the term “substantial absence” refers to a level of anactivating or inhibiting factor that is below the level necessary toactivate or inhibit GPCR function by at least 10% as measured by a givenassay disclosed herein or known in the art.

As used herein, the term “G-Protein coupled receptor,” or “GPCR” refersto a membrane-associated polypeptide with 7 alpha helical transmembranedomains. Functional GPCR's associate with a ligand or agonist and alsoassociate with and activate G-proteins. ChemerinR is a GPCR.

As used herein, the term “agent that modulates the function of aChemerinR polypeptide” is a molecule or compound that increases ordecreases ChemerinR activity, including compounds that change thebinding of Chemerin polypeptides or other agonists, and compounds thatchange ChemerinR downstream signaling activities.

As used herein, the term “null mutation” refers to an insertion,deletion, or substitution that modifies the chromosomal sequencesencoding a polypeptide, such that the polypeptide is not expressed.

I. Assays For The Identification of Agents that Modulate the Activity ofChemerinR

Agents that modulate the activity of ChemerinR can be identified in anumber of ways that take advantage of the interaction of the receptorwith Chemerin. For example, the ability to reconstituteChemerinR/Chemerin binding either in vitro, on cultured cells or in vivoprovides a target for the identification of agents that disrupt thatbinding. Assays based on disruption of binding can identify agents, suchas small organic molecules, from libraries or collections of suchmolecules. Alternatively, such assays can identify agents in samples orextracts from natural sources, e.g., plant, fungal or bacterial extractsor even in human tissue samples (e.g., tumor tissue). In one aspect, theextracts can be made from cells expressing a library of variant nucleicacids, peptides or polypeptides, including, for example, variants ofChemerin polypeptide itself. Modulators of ChemerinR/Chemerin bindingcan then be screened using a binding assay or a functional assay thatmeasures downstream signaling through the receptor. Both binding assaysand functional assays are validated using Chemerin polypeptide.

Another approach that uses the ChemerinR/Chemerin interaction moredirectly to identify agents that modulate ChemerinR function measureschanges in ChemerinR downstream signaling induced by candidate agents orcandidate modulators. These functional assays can be performed inisolated cell membrane fractions or on cells expressing the receptor ontheir surfaces.

A. ChemerinR Polypeptides.

Assays using the interaction of ChemerinR and Chemerin require a sourceof ChemerinR polypeptide. The polynucleotide and polypeptide sequence ofhuman ChemerinR are presented herein as SEQ ID NOs: 1 and 2. The humanChemerinR polynucleotide sequence is also available at GenBank AccessionNo. Y14838, and was reported in Samson et al., 1998, Eur. J. Immunol.28: 1689-1700, incorporated herein by reference. ChemerinR polypeptidesequence is also recorded at accession Nos. O75748 and CAA75112 in theSwissprot database. Related sequences include those for CMKRL1 (GenBankAccession Nos. XM_(—)006864 and NM004072 (nucleotide sequences) andSwissprot Accession No. Q99788 (polypeptide sequence)), human DEZb(GenBank Accession No. U79527 (nucleotide sequence)), human DEZa(GenBank Accession No. U79526 (nucleotide sequence), mouse DEZ (GenBankAccession No. U79525 (nucleotide sequence) and Swissprot Accession No.P97468 (polypeptide sequence)), and rat ChemerinR (GenBank Accession No.AJ002745 (nucleotide sequence) and Swissprot Accession No. O35786(polypeptide sequence).

One skilled in the art can readily amplify a ChemerinR sequence from asample containing mRNA encoding the protein through basic PCR andmolecular cloning techniques using primers or probes designed from theknown sequences.

The expression of recombinant polypeptides is well known in the art.Those skilled in the art can readily select vectors and expressioncontrol sequences for the expression of ChemerinR polypeptides usefulaccording to the invention in eukaryotic or prokaryotic cells. ChemerinRmust be associated with cell membrane or detergents like syntheticliposomes in order to have binding or signaling function. Methods forthe preparation of cellular membrane fractions are well known in theart, e.g., the method reported by Hubbard & Cohn, 1975, J. Cell Biol.64: 461-479, which is incorporated herein by reference. In order toproduce membranes comprising ChemerinR, one need only apply suchtechniques to cells endogenously or recombinantly expressing ChemerinR.Alternatively, membrane-free ChemerinR can be integrated into membranepreparations by dilution of detergent solution of the polypeptide (see,e.g., Salamon et al., 1996, Biophys. J. 71:283-294, which isincorporated herein by reference).

B. Chemerin Polypeptides.

The present invention relates to a Chemerin polypeptide including thefull-length active form and the truncated Chemerin polypeptides. The 163amino acid full-length Preprochemerin polypeptide is first produced in acell as inactive form (FIG. 6). This inactive form of Chemerin isconverted into the active form of Chemerin (137 amino acids) by thefollowing two steps: a) removing 20 amino acids at N-terminus (this formis called prochemerin, 143 amino acids, FIG. 8); b) removing 6 aminoacids at C-terminus (137 amino acids, FIG. 9). Preferably, theC-terminus human truncated Preprochemerin and chemerin polypeptides arepresented in FIGS. 16, 8 and 20 a (human chemerin-9, -10, -11, -12, -13,-19) respectively. The Chemerin polypeptides of the invention may be arecombinant Chemerin polypeptide, a natural Chemerin polypeptide, or asynthetic Chemerin polypeptide, preferably a recombinant Chemerinpolypeptide. The Chemerin polypeptide of the invention may alsoencompass the analogs or variants whose polypeptide sequences aredifferent from the naturally-occurring ones, but retain substantiallythe same function or activity as a Chemerin polypeptide.

The full-length human inactive Preprochemerin polynucleotide andpolypeptide sequences are presented herein as SEQ ID Nos 7 and 8,respectively (FIG. 6). Preprochemerin sequences are also available fromGenBank (e.g., Human polynucleotide sequences include Accession Nos. XM004765, U77594, NM 002889, human polypeptide sequence is available atAccession Nos. Q99969, BAA76499, AAB47975, NP002880, and XP004765;Gallus gallus polynucleotide sequences include Accession Nos. BG713704,BG713660 and BG713614; mouse polynucleotide sequences include BF020273,AW113641 and bf018000; rat polynucleotide sequences include AW915104;Sus scrofa polynucleotide sequences include BF078978 and BF713092(overlapping ESTs, last 7 amino acids of Preprochemerin sequence inBF713092); and Bos taurus polynucleotide sequences include BG691132). Analignment of Preprochemerin sequences is presented in FIG. 11.

The present invention also relates to a nucleic acid sequence thatencodes a Chemerin polypeptide. The nucleic acid sequences of theinvention may also contain the coding sequences fused in frame to amarker sequence for purification of the polypeptides of the presentinvention. The nucleic acid sequences of the present invention may beemployed for producing polypeptides of the present invention byrecombinant techniques. The nucleic acid sequences of the invention maybe included in any one of the expressing vectors such as plasmid DNA,phage DNA, or Viral DNA vectors etc, all vectors are well known in theart.

As with ChemerinR, Chemerin polynucleotides can be cloned throughstandard PCR and molecular cloning techniques using the known sequencesas a source of amplification primers or probes. Similarly, clonedChemerin polypeptides can be expressed in eukaryotic or prokaryoticcells as known in the art. As a non-limiting example, Chemerin may becloned into an acceptable mammalian expression vector, such as pCDNA3(Invitrogen) for expression in a host cell. A Chemerin expressionconstruct for expression in yeast is described in Example 4.

Chemerin can also be expressed in vitro through in vitro transcriptionand translation. Further, if desired for a given assay or technique,Chemerin polypeptides useful according to the invention can be producedas fusion proteins or tagged proteins. For example, either full lengthChemerin or a portion thereof (i.e., at least 10 amino acids, preferablyat least 20 amino acids or more, up to one amino acid less than fulllength Chemerin) can be fused to Glutathione-S-Transferase (GST),secreted alkaline phosphatase (SEAP), a FLAG tag, a Myc tag, or a 6X-Hispeptide to facilitate the purification or detection of the Chemerinpolypeptide. Methods and vectors for the production of tagged or fusionproteins are well known in the art, as are methods of isolating anddetecting such fused or tagged proteins.

Recombinant Chemerin polypeptides can be used in purified form.Alternatively, conditioned medium from Chemerin transfected cells can beused. The amounts of Chemerin necessary in a given binding or functionalassay according to the invention will vary depending upon the assay, butwill generally use 1 pM to 1 nM of labeled and 10 pM to 1 μM ofunlabeled Chemerin per assay. The affinities and EC₅₀s of taggedChemerin polypeptides for ChemerinR may vary relative to those of fulllength wild type Chemerin polypeptide, and the amount necessary for agiven assay can therefore be adjusted relative to the wild-type values.If necessary for a given assay, Chemerin can be labeled by incorporationof radiolabeled amino acids in the medium during synthesis, e.g.,³⁵S-Met, ¹⁴C-Leu, tritium H3 or others as appropriate. Methods ofchemical labeling with ¹²⁵I are known in the art. Fluorescent labels canalso be attached to Chemerin polypeptides or to other ChemerinR ligandsusing standard labeling techniques.

The Chemerin polypeptides may also be employed for treatment of adisease or disorder. For example, cells from a patient may be engineeredwith a polynucleotide (DNA or RNA) encoding a polypeptide ex vivo, withthe engineered cells then being provided to a patient to be treated withthe polypeptide. Such methods are well-known in the art. Similarly,cells may be engineered in vivo for expression of a polypeptide in vivoby, for example, procedures known in the art. These and other methodsfor administering a polypeptide of the present invention by such methodshould be apparent to those skilled in the art from the teachings of thepresent invention. For example, the expression vector for engineeringcells in vivo may be a retrovirus, an adenovirus, or a non-viralvectors.

C. Assays to Identify Modulators of ChemerinR Activity

The discovery that Chemerin is a ligand of the ChemerinR receptorpermits screening assays to identify agonists, antagonists and inverseagonists of receptor activity. The screening assays will have twogeneral approaches.

1) Ligand binding assays, in which cells expressing ChemerinR, membraneextracts from such cells, or immobilized lipid membranes comprisingChemerinR are exposed to a labeled Chemerin polypeptide and candidatecompound. Following incubation, the reaction mixture is measured forspecific binding of the labeled Chemerin polypeptide to the ChemerinRreceptor. Compounds that interfere with or displace labeled Chemerinpolypeptide can be agonists, antagonists or inverse agonists ofChemerinR activity. Functional analysis can be performed on positivecompounds to determine which of these categories they fit.

2) Functional assays, in which a signaling activity of ChemerinR ismeasured.

a) For agonist screening, cells expressing ChemerinR or membranesprepared from them are incubated with candidate compound, and asignaling activity of ChemerinR is measured. The assays are validatedusing a Chemerin polypeptide as agonist, and the activity induced bycompounds that modulate receptor activity is compared to that induced byChemerin. An agonist or partial agonist will have a maximal biologicalactivity corresponding to at least 10% of the maximal activity of wildtype human Chemerin when the agonist or partial agonist is present at 10μM or less, and preferably will have 50%, 75%, 100% or more, including2-fold, 5-fold, 10-fold or more activity than wild-type human Chemerin.

b) For antagonist or inverse agonist screening, cells expressingChemerinR or membranes isolated from them are assayed for signalingactivity in the presence of a Chemerin polypeptide with or without acandidate compound. Antagonists or inverse agonists will reduce thelevel of Chemerin-stimulated receptor activity by at least 10%, relativeto reactions lacking the antagonist or inverse agonist.

c) For inverse agonist screening, cells expressing constitutiveChemerinR activity or membranes isolated from them are used in afunctional assay that measures an activity of the receptor in thepresence and absence of a candidate compound. Inverse agonists are thosecompounds that reduce the constitutive activity of the receptor by atleast 10%. Overexpression of ChemerinR (i.e., expression of 5-fold orhigher excess of ChemerinR polypeptide relative to the level naturallyexpressed in macro phages in vivo) may lead to constitutive activation.ChememerinR can be overexpressed by placing it under the control of astrong constitutive promoter, e.g., the CMV early promoter.Alternatively, certain mutations of conserved GPCR amino acids or aminoacid domains tend to lead to constitutive activity. See for example:Kjelsberg et al., 1992, J. Biol. Chem. 267:1430; McWhinney et al., 2000.J. Biol. Chem. 275:2087; Ren et al., 1993, J. Biol. Chem. 268:16483;Samama et al., 1993, J. Biol. Chem. 268:4625; Parma et al., 1993, Nature365:649; Parma et al., 1998, J. Pharmacol. Exp. Ther. 286:85; and Parentet al., 1996, J. Biol. Chem. 271:7949.

Ligand Binding and Displacement Assays:

One can use ChemerinR polypeptides expressed on a cell, or isolatedmembranes containing receptor polypeptides, along with a Chemerinpolypeptide in order to screen for compounds that inhibit the binding ofChemerin to ChemerinR. When identified in an assay that measures bindingor Chemerin polypeptide displacement alone, compounds will have to besubjected to functional testing to determine whether they act asagonists, antagonists or inverse agonists.

For displacement experiments, cells expressing a ChemerinR polypeptide(generally 25,000 cells per assay or 1 to 100 μg of membrane extracts)are incubated in binding buffer (e.g., 50 mM Hepes pH 7.4; 1 mM CaCl₂;0.5% Bovine Serum Albumin (BSA) Fatty Acid-Free; and 0 5 mM MgCl 2) for1.5 hrs (at, for example, 27° C.) with labeled Chemerin polypeptide inthe presence or absence of increasing concentrations of a candidatemodulator. To validate and calibrate the assay, control competitionreactions using increasing concentrations of unlabeled Chemerinpolypeptide can be performed. After incubation, cells are washedextensively, and bound, labeled Chemerin is measured as appropriate forthe given label (e.g., scintillation counting, enzyme assay,fluorescence, etc.). A decrease of at least 10% in the amount of labeledChemerin polypeptide bound in the presence of candidate modulatorindicates displacement of binding by the candidate modulator. Candidatemodulators are considered to bind specifically in this or other assaysdescribed herein if they displace 50% of labeled Chemerin(sub-saturating Chemerin dose) at a concentration of 10 μM or less(i.e., EC₅₀ is 10 μM or less).

Alternatively, binding or displacement of binding can be monitored bysurface plasmon resonance (SPR). Surface plasmon resonance assays can beused as a quantitative method to measure binding between two moleculesby the change in mass near an immobilized sensor caused by the bindingor loss of binding of a Chemerin polypeptide from the aqueous phase to aChemerinR polypeptide immobilized in a membrane on the sensor. Thischange in mass is measured as resonance units versus time afterinjection or removal of the Chemerin polypeptide or candidate modulatorand is measured using a Biacore Biosensor (Biacore AB). ChemerinR can beimmobilized on a sensor chip (for example, research grade CM5 chip;Biacore AB) in a thin film lipid membrane according to methods describedby Salamon et al. (Salamon et al., 1996, Biophys J. 71: 283-294; Salamonet al., 2001, Biophys. J. 80: 1557-1567; Salamon et al., 1999, TrendsBiochem. Sci. 24: 213-219, each of which is incorporated herein byreference.). Sarrio et al. demonstrated that SPR can be used to detectligand binding to the GPCR A(1) adenosine receptor immobilized in alipid layer on the chip (Sarrio et al., 2000, Mol. Cell. Biol. 20:5164-5174, incorporated herein by reference). Conditions for Chemerinbinding to ChemerinR in an SPR assay can be fine-tuned by one of skillin the art using the conditions reported by Sarrio et al. as a startingpoint.

SPR can assay for modulators of binding in at least two ways. First, aChemerin polypeptide can be pre-bound to immobilized ChemerinRpolypeptide, followed by injection of candidate modulator atapproximately 10 μl/min flow rate and a concentration ranging from 1 nMto 100 μM, preferably about 1 μM. Displacement of the bound Chemerin canbe quantitated, permitting detection of modulator binding.Alternatively, the membrane-bound ChemerinR polypeptide can bepre-incubated with candidate modulator and challenged with a Chemerinpolypeptide. A difference in Chemerin binding to the ChemerinR exposedto modulator relative to that on a chip not pre-exposed to modulatorwill demonstrate binding. In either assay, a decrease of 10% or more inthe amount of a Chemerin polypeptide bound is in the presence ofcandidate modulator, relative to the amount of a Chemerin polypeptidebound in the absence of candidate modulator indicates that the candidatemodulator inhibits the interaction of ChemerinR and Chemerin.

Another method of measuring inhibition of binding of a Chemerinpolypeptide to ChemerinR uses fluorescence resonance energy transfer(FRET). FRET is a quantum mechanical phenomenon that occurs between afluorescence donor (D) and a fluorescence acceptor (A) in closeproximity to each other (usually <100 A of separation) if the emissionspectrum of D overlaps with the excitation spectrum of A. The moleculesto be tested, e.g., a Chemerin polypeptide and a ChemerinR polypeptide,are labeled with a complementary pair of donor and acceptorfluorophores. While bound closely together by the ChemerinR:Chemerininteraction, the fluorescence emitted upon excitation of the donorfluorophore will have a different wavelength than that emitted inresponse to that excitation wavelength when the polypeptides are notbound, providing for quantitation of bound versus unbound polypeptidesby measurement of emission intensity at each wavelength. Donor:Acceptorpairs of fluorophores with which to label the polypeptides are wellknown in the art. Of particular interest are variants of the A. VictoriaGFP known as Cyan FP (CFP, Donor(D)) and Yellow FP(YFP, Acceptor(A)).The GFP variants can be made as fusion proteins with the respectivemembers of the binding pair to serve as D-A pairs in a FRET scheme tomeasure protein-protein interaction. Vectors for the expression of GFPvariants as fusions are known in the art. As an example, a CFP-Chemerinfusion and a YFP-ChemerinR fusion can be made. The addition of acandidate modulator to the mixture of labeled Chemerin and ChemerinRproteins will result in an inhibition of energy transfer evidenced by,for example, a decrease in YFP fluorescence relative to a sample withoutthe candidate modulator. In an assay using FRET for the detection ofChemerinR:Chemerin interaction, a 10% or greater decrease in theintensity of fluorescent emission at the acceptor wavelength in samplescontaining a candidate modulator, relative to samples without thecandidate modulator, indicates that the candidate modulator inhibitsChemerinR:Chemerin interaction.

A variation on FRET uses fluorescence quenching to monitor molecularinteractions. One molecule in the interacting pair can be labeled with afluorophore, and the other with a molecule that quenches thefluorescence of the fluorophore when brought into close apposition withit. A change in fluorescence upon excitation is indicative of a changein the association of the molecules tagged with the fluorophore:quencherpair. Generally, an increase in fluorescence of the labeled ChemerinRpolypeptide is indicative that the Chemerin polypeptide bearing thequencher has been displaced. For quenching assays, a 10% or greaterincrease in the intensity of fluorescent emission in samples containinga candidate modulator, relative to samples without the candidatemodulator, indicates that the candidate modulator inhibitsChemerinR:Chemerin interaction.

In addition to the surface plasmon resonance and FRET methods,fluorescence polarization measurement is useful to quantitateprotein-protein binding. The fluorescence polarization value for afluorescently-tagged molecule depends on the rotational correlation timeor tumbling rate. Protein complexes, such as those formed by ChemerinRassociating with a fluorescently labeled Chemerin polypeptide, havehigher polarization values than uncomplexed, labeled Chemerin. Theinclusion of a candidate inhibitor of the ChemerinR:Chemerin interactionresults in a decrease in fluorescence polarization, relative to amixture without the candidate inhibitor, if the candidate inhibitordisrupts or inhibits the interaction of ChemerinR with Chemerin.Fluorescence polarization is well suited for the identification of smallmolecules that disrupt the formation of polypeptide or proteincomplexes. A decrease of 10% or more in fluorescence polarization insamples containing a candidate modulator, relative to fluorescencepolarization in a sample lacking the candidate modulator, indicates thatthe candidate modulator inhibits ChemerinR:Chemerin interaction.

Another alternative for monitoring ChemerinR:Chemerin interactions usesa biosensor assay. ICS biosensors have been described by AMBRI(Australian Membrane Biotechnology Research Institute;http//www.ambri.com.au/). In this technology, the association ofmacromolecules such as ChemerinR and Chemerin, is coupled to the closingof gramacidin-facilitated ion channels in suspended membrane bilayersand thus to a measurable change in the admittance (similar to impedance)of the biosensor. This approach is linear over six orders of magnitudeof admittance change and is ideally suited for large scale, highthroughput screening of small molecule combinatorial libraries. A 10% orgreater change (increase or decrease) in admittance in a samplecontaining a candidate modulator, relative to the admittance of a samplelacking the candidate modulator, indicates that the candidate modulatorinhibits the interaction of ChemerinR and Chemerin.

It is important to note that in assays of protein-protein interaction,it is possible that a modulator of the interaction need not necessarilyinteract directly with the domain(s) of the proteins that physicallyinteract. It is also possible that a modulator will interact at alocation removed from the site of protein-protein interaction and cause,for example, a conformational change in the ChemerinR polypeptide.Modulators (inhibitors or agonists) that act in this manner arenonetheless of interest as agents to modulate the activity of ChemerinR.

It should be understood that any of the binding assays described hereincan be performed with a non-Chemerin ligand (for example, agonist,antagonist, etc.) of ChemerinR, e.g., a small molecule identified asdescribed herein. In practice, the use of a small molecule ligand orother non-Chemerin ligand has the benefit that non-polypeptide chemicalcompounds are generally cheaper and easier to produce in purified formthan polypeptides such as Chemerin. Thus, a non-Chemerin ligand isbetter suited to high-throughput assays for the identification ofagonists, antagonists or inverse agonists than full length Chemerin.This advantage in no way erodes the importance of assays using Chemerin,however, as such assays are well suited for the initial identificationof non-Chemerin ligands.

Any of the binding assays described can be used to determine thepresence of an agent in a sample, e.g., a tissue sample, that binds tothe ChemerinR receptor molecule, or that affects the binding of Chemerinto the receptor. To do so, ChemerinR polypeptide is reacted withChemerin polypeptide or another ligand in the presence or absence of thesample, and Chemerin or ligand binding is measured as appropriate forthe binding assay being used. A decrease of 10% or more in the bindingof Chemerin or other ligand indicates that the sample contains an agentthat modulates Chemerin or ligand binding to the receptor polypeptide.

Functional Assays of Receptor Activity

i. GTPase/GTP Binding Assays:

For GPCRs such as ChemerinR, a measure of receptor activity is thebinding of GTP by cell membranes containing receptors. In the methoddescribed by Traynor and Nahorski, 1995, Mol. Pharmacol. 47: 848-854,incorporated herein by reference, one essentially measures G-proteincoupling to membranes by measuring the binding of labeled GTP. For GTPbinding assays, membranes isolated from cells expressing the receptorare incubated in a buffer containing 20 mM HEPES, pH 7.4, 100 mM NaCl,and 10 mM MgCl2, 80 pM ³⁵S-GTPγS and 3 μM GDP. The assay mixture isincubated for 60 minutes at 30° C., after which unbound labeled GTP isremoved by filtration onto GF/B filters. Bound, labeled GTP is measuredby liquid scintillation counting. In order to assay for modulation ofChemerin-induced ChemerinR activity, membranes prepared from cellsexpressing a ChemerinR polypeptide are mixed with a Chemerinpolypeptide, and the GTP binding assay is performed in the presence andabsence of a candidate modulator of ChemerinR activity. A decrease of10% or more in labeled GTP binding as measured by scintillation countingin an assay of this kind containing candidate modulator, relative to anassay without the modulator, indicates that the candidate modulatorinhibits ChemerinR activity.

A similar GTP-binding assay can be performed without Chemerin toidentify compounds that act as agonists. In this case,Chemerin-stimulated GTP binding is used as a standard. A compound isconsidered an agonist if it induces at least 50% of the level of GTPbinding induced by full length wild-type Chemerin when the compound ispresent at 1 μM or less, and preferably will induce a level the same asor higher than that induced by Chemerin.

GTPase activity is measured by incubating the membranes containing aChemerinR polypeptide with γ³²P-GTP. Active GTPase will release thelabel as inorganic phosphate, which is detected by separation of freeinorganic phosphate in a 5% suspension of activated charcoal in 20 mMH₃PO₄, followed by scintillation counting. Controls include assays usingmembranes isolated from cells not expressing ChemerinR(mock-transfected), in order to exclude possible non-specific effects ofthe candidate compound.

In order to assay for the effect of a candidate modulator onChemerinR-regulated GTPase activity, membrane samples are incubated witha Chemerin polypeptide, with and without the modulator, followed by theGTPase assay. A change (increase or decrease) of 10% or more in thelevel of GTP binding or GTPase activity relative to samples withoutmodulator is indicative of ChemerinR modulation by a candidatemodulator.

ii. Downstream Pathway Activation Assays:

a. Calcium Flux—The Aequorin-Based Assay.

The aequorin assay takes advantage of the responsiveness ofmitochondrial apoaequorin to intracellular calcium release induced bythe activation of GPCRs (Stables et al., 1997, Anal. Biochem.252:115-126; Detheux et al., 2000, J. Exp. Med., 192 1501-1508; both ofwhich are incorporated herein by reference). Briefly,ChemerinR-expressing clones are transfected to coexpress mitochondrialapoaequorin and Gα16. Cells are incubated with 5 μM Coelenterazine H(Molecular Probes) for 4 hours at room temperature, washed in DMEM-F12culture medium and resuspended at a concentration of 0.5×10⁶ cells/ml.Cells are then mixed with test agonist peptides and light emission bythe aequorin is recorded with a luminometer for 30 sec. Results areexpressed as Relative Light Units (RLU). Controls include assays usingmembranes isolated from cells not expressing ChemerinR(mock-transfected), in order to exclude possible non-specific effects ofthe candidate compound.

Aequorin activity or intracellular calcium levels are “changed” if lightintensity increases or decreases by 10% or more in a sample of cells,expressing a ChemerinR polypeptide and treated with a candidatemodulator, relative to a sample of cells expressing the ChemerinRpolypeptide but not treated with the candidate modulator or relative toa sample of cells not expressing the ChemerinR polypeptide(mock-transfected cells) but treated with the candidate modulator.

When performed in the absence of a Chemerin polypeptide, the assay canbe used to identify an agonist of ChemerinR activity. When the assay isperformed in the presence of a Chemerin polypeptide, it can be used toassay for an antagonist.

b. Adenylate Cyclase Assay:

Assays for adenylate cyclase activity are described by Kenimer &Nirenberg, 1981, Mol. Pharmacol. 20: 585-591, incorporated herein byreference. That assay is a modification of the assay taught by Solomonet al., 1974, Anal. Biochem. 58: 541-548, also incorporated herein byreference. Briefly, 100 μl reactions contain 50 mM Tris-Hcl (pH 7.5), 5mM MgCl₂, 20 mM creatine phosphate (disodium salt), 10 units (71 μg ofprotein) of creatine phosphokinase, 1 mM α-³²P-ATP (tetrasodium salt, 2μCi), 0.5 mM cyclic AMP, G-³H-labeled cyclic AMP (approximately 10,000cpm), 0.5 mM Ro20-1724, 0.25% ethanol, and 50-200 μg of proteinhomogenate to be tested (i.e., homogenate from cells expressing or notexpressing a ChemerinR polypeptide, treated or not treated with aChemerin polypeptide with or without a candidate modulator). Reactionmixtures are generally incubated at 37° C. for 6 minutes. Followingincubation, reaction mixtures are deproteinized by the addition of 0.9ml of cold 6% trichloroacetic acid. Tubes are centrifuged at 1800×g for20 minutes and each supernatant solution is added to a Dowex AG50W-X4column. The cAMP fraction from the column is eluted with 4 ml of 0.1 mMimidazole-HCl (pH 7.5) into a counting vial. Assays should be performedin triplicate. Control reactions should also be performed using proteinhomogenate from cells that do not express a ChemerinR polypeptide.

According to the invention, adenylate cyclase activity is “changed” ifit increases or decreases by 10% or more in a sample taken from cellstreated with a candidate modulator of ChemerinR activity, relative to asimilar sample of cells not treated with the candidate modulator orrelative to a sample of cells not expressing the ChemerinR polypeptide(mock-transfected cells) but treated with the candidate modulator.

c. cAMP Assay:

Intracellular or extracellular cAMP is measured using a cAMPradioimmunoassay (RIA) or cAMP binding protein according to methodswidely known in the art. For example, Horton & Baxendale, 1995, MethodsMol. Biol. 41: 91-105, which is incorporated herein by reference,describes an RIA for cAMP.

A number of kits for the measurement of cAMP are commercially available,such as the High Efficiency Fluorescence Polarization-based homogeneousassay marketed by LJL Biosystems and NEN Life Science Products. Controlreactions should be performed using extracts of mock-transfected cellsto exclude possible non-specific effects of some candidate modulators.

The level of cAMP is “changed” if the level of cAMP detected in cells,expressing a ChemerinR polypeptide and treated with a candidatemodulator of ChemerinR activity (or in extracts of such cells), usingthe RIA-based assay of Horton & Baxendale, 1995, supra, increases ordecreases by at least 10% relative to the cAMP level in similar cellsnot treated with the candidate modulator.

d. Phospholipid Breakdown, DAG Production and Inositol TriphosphateLevels:

Receptors that activate the breakdown of phospholipids can be monitoredfor changes due to the activity of known or suspected modulators ofChemerinR by monitoring phospholipid breakdown, and the resultingproduction of second messengers DAG and/or inositol triphosphate (IP3).Methods of measuring each of these are described in PhospholipidSignaling Protocols, edited by Ian M. Bird. Totowa, N.J., Humana Press,1998, which is incorporated herein by reference. See also Rudolph etal., 1999, J. Biol. Chem. 274: 11824-11831, incorporated herein byreference, which also describes an assay for phosphatidylinositolbreakdown. Assays should be performed using cells or extracts of cellsexpressing ChemerinR, treated or not treated with a Chemerin polypeptidewith or without a candidate modulator. Control reactions should beperformed using mock-transfected cells, or extracts from them in orderto exclude possible non-specific effects of some candidate modulators.

According to the invention, phosphatidylinositol breakdown, anddiacylglycerol and/or inositol triphosphate levels are “changed” if theyincrease or decrease by at least 10% in a sample from cells expressing aChemerinR polypeptide and treated with a candidate modulator, relativeto the level observed in a sample from cells expressing a ChemerinRpolypeptide that is not treated with the candidate modulator.

e. PKC Activation Assays:

Growth factor receptor tyrosine kinases tend to signal via a pathwayinvolving activation of Protein Kinase C(PKC), which is a family ofphospholipid- and calcium-activated protein kinases. PKC activationultimately results in the transcription of an array of proto-oncogenetranscription factor-encoding genes, including c-fos, c-myc and c-jun,proteases, protease inhibitors, including collagenase type I andplasminogen activator inhibitor, and adhesion molecules, includingintracellular adhesion molecule I (ICAM I). Assays designed to detectincreases in gene products induced by PKC can be used to monitor PKCactivation and thereby receptor activity. In addition, the activity ofreceptors that signal via PKC can be monitored through the use ofreporter gene constructs driven by the control sequences of genesactivated by PKC activation. This type of reporter gene-based assay isdiscussed in more detail below.

For a more direct measure of PKC activity, the method of Kikkawa et al.,1982, J. Biol. Chem. 257: 13341, incorporated herein by reference, canbe used. This assay measures phosphorylation of a PKC substrate peptide,which is subsequently separated by binding to phosphocellulose paper.This PKC assay system can be used to measure activity of purifiedkinase, or the activity in crude cellular extracts. Protein kinase Csample can be diluted in 20 mM HEPES/2 mM DTT immediately prior toassay.

The substrate for the assay is the peptide Ac-FKKSFKL-NH2 (SEQ ID NO:80), derived from the myristoylated alanine-rich protein kinase Csubstrate protein (MARCKS). The K_(m) of the enzyme for this peptide isapproximately 50 μM. Other basic, protein kinase C-selective peptidesknown in the art can also be used, at a concentration of at least 2-3times their K_(m). Cofactors required for the assay include calcium,magnesium, ATP, phosphatidylserine and diacylglycerol. Depending uponthe intent of the user, the assay can be performed to determine theamount of PKC present (activating conditions) or the amount of activePCK present (non-activating conditions). For most purposes according tothe invention, non-activating conditions will be used, such that the PKCthat is active in the sample when it is isolated is measured, ratherthan measuring the PKC that can be activated. For non-activatingconditions, calcium is omitted in the assay in favor of EGTA.

The assay is performed in a mixture containing 20 mM HEPES, pH 7.4, 1-2mM DTT, 5 mM MgCl₂, 100 μM ATP, ˜1 μCi γ-³²P-ATP, 100 μg/ml peptidesubstrate (˜100 μM), 140 μM/3.8 μM phosphatidylserine/diacylglycerolmembranes, and 100 μM calcium (or 500 μM EGTA). 48 μl of sample, dilutedin 20 mM HEPES, pH 7.4, 2 mM DTT is used in a final reaction volume of80 μl. Reactions are performed at 30° C. for 5-10 minutes, followed byaddition of 25 μl of 100 mM ATP, 100 mM EDTA, pH 8.0, which stops thereactions.

After the reaction is stopped, a portion (85 μl) of each reaction isspotted onto a Whatman P81 cellulose phosphate filter, followed bywashes: four times 500 ml in 0.4% phosphoric acid, (5-10 min per wash);and a final wash in 500 ml 95% EtOH, for 2-5 min. Bound radioactivity ismeasured by scintillation counting. Specific activity (cpm/nmol) of thelabeled ATP is determined by spotting a sample of the reaction onto P81paper and counting without washing. Units of PKC activity, defined asnmol phosphate transferred per min, are calculated as follows:

The activity, in UNITS (nmol/min) is:

$= {\frac{\left( {{cpm}\mspace{14mu} {on}\mspace{14mu} {paper}} \right) \times \left( {105\mspace{14mu} {\mu l}\mspace{14mu} {total}\text{/}85\mspace{14mu} {\mu l}\mspace{14mu} {spotted}} \right)}{\left( {{{assay}\mspace{14mu} {time}},\min} \right)\left( {{specific}\mspace{14mu} {activity}\mspace{14mu} {of}\mspace{14mu} {ATP}\mspace{14mu} {cpm}\text{/}{nmol}} \right)}.}$

An alternative assay can be performed using a Protein Kinase C Assay Kitsold by PanVera (Cat. # P2747).

Assays are performed on extracts from cells expressing a ChemerinRpolypeptide, treated or not treated with a Chemerin polypeptide with orwithout a candidate modulator. Control reactions should be performedusing mock-transfected cells, or extracts from them in order to excludepossible non-specific effects of some candidate modulators.

According to the invention, PKC activity is “changed” by a candidatemodulator when the units of PKC measured by either assay described aboveincrease or decrease by at least 10%, in extracts from cells expressingChemerinR and treated with a candidate modulator, relative to a reactionperformed on a similar sample from cells not treated with a candidatemodulator.

f. Kinase Assays:

MAP kinase activity can be assayed using any of several kits availablecommercially, for example, the p38 MAP Kinase assay kit sold by NewEngland Biolabs (Cat # 9820) or the FlashPlate™ MAP Kinase assays soldby Perkin-Elmer Life Sciences.

MAP Kinase activity is “changed” if the level of activity is increasedor decreased by 10% or more in a sample from cells, expressing aChemerinR polypeptide, treated with a candidate modulator relative toMAP kinase activity in a sample from similar cells not treated with thecandidate modulator.

Direct assays for tyrosine kinase activity using known synthetic ornatural tyrosine kinase substrates and labeled phosphate are well known,as are similar assays for other types of kinases (e.g., Ser/Thrkinases). Kinase assays can be performed with both purified kinases andcrude extracts prepared from cells expressing a ChemerinR polypeptide,treated with or without a Chemerin polypeptide, with or without acandidate modulator. Control reactions should be performed usingmock-transfected cells, or extracts from them in order to excludepossible non-specific effects of some candidate modulators. Substratescan be either full length protein or synthetic peptides representing thesubstrate. Pinna & Ruzzene (1996, Biochem. Biophys. Acta 1314: 191-225,incorporated herein by reference) list a number of phosphorylationsubstrate sites useful for measuring kinase activities. A number ofkinase substrate peptides are commercially available. One that isparticularly useful is the “Src-related peptide,” RRLIEDAEYAARG (SEQ IDNO: 74; available from Sigma # A7433), which is a substrate for manyreceptor and nonreceptor tyrosine kinases. Because the assay describedbelow requires binding of peptide substrates to filters, the peptidesubstrates should have a net positive charge to facilitate binding.Generally, peptide substrates should have at least 2 basic residues anda free amino terminus. Reactions generally use a peptide concentrationof 0.7-1.5 mM.

Assays are generally carried out in a 25 μl volume comprising 5 μl of 5×kinase buffer (5 mg/mL BSA, 150 mM Tris-Cl (pH 7.5), 100 mM MgCl₂;depending upon the exact kinase assayed for, MnCl₂ can be used in placeof or in addition to the MgCl₂), 5 μl of 1.0 mM ATP (0.2 mM finalconcentration), γ-³²P-ATP (100-500 cpm/μmol), 3 μl of 10 mM peptidesubstrate (1.2 mM final concentration), cell extract containing kinaseto be tested (cell extracts used for kinase assays should contain aphosphatase inhibitor (e.g. 0.1-1 mM sodium orthovanadate)), and H₂O to25 μl. Reactions are performed at 30° C., and are initiated by theaddition of the cell extract.

Kinase reactions are performed for 30 seconds to about 30 minutes,followed by the addition of 45 μl of ice-cold 10% trichloroacetic acid(TCA). Samples are spun for 2 minutes in a microcentrifuge, and 35 μl ofthe supernatant is spotted onto Whatman P81 cellulose phosphate filtercircles. The filters are washed three times with 500 ml cold 0.5%phosphoric acid, followed by one wash with 200 ml of acetone at roomtemperature for 5 minutes. Filters are dried and incorporated 32P ismeasured by scintillation counting. The specific activity of ATP in thekinase reaction (e.g., in cpm/pmol) is determined by spotting a smallsample (2-5 μl) of the reaction onto a P81 filter circle and countingdirectly, without washing. Counts per minute obtained in the kinasereaction (minus blank) are then divided by the specific activity todetermine the moles of phosphate transferred in the reaction.

Tyrosine kinase activity is “changed” if the level of kinase activity isincreased or decreased by 10% or more in a sample from cells, expressinga ChemerinR polypeptide, treated with a candidate modulator relative tokinase activity in a sample from similar cells not treated with thecandidate modulator.

g. Transcriptional Reporters for Downstream Pathway Activation:

The intracellular signal initiated by binding of an agonist to areceptor, e.g., ChemerinR, sets in motion a cascade of intracellularevents, the ultimate consequence of which is a rapid and detectablechange in the transcription or translation of one or more genes. Theactivity of the receptor can therefore be monitored by measuring theexpression of a reporter gene driven by control sequences responsive toChemerinR activation.

As used herein “promoter” refers to the transcriptional control elementsnecessary for receptor-mediated regulation of gene expression, includingnot only the basal promoter, but also any enhancers ortranscription-factor binding sites necessary for receptor-regulatedexpression. By selecting promoters that are responsive to theintracellular signals resulting from agonist binding, and operativelylinking the selected promoters to reporter genes whose transcription,translation or ultimate activity is readily detectable and measurable,the transcription based reporter assay provides a rapid indication ofwhether a given receptor is activated.

Reporter genes such as luciferase, CAT, GFP, β-lactamase orβ-galactosidase are well known in the art, as are assays for thedetection of their products.

Genes particularly well suited for monitoring receptor activity are the“immediate early” genes, which are rapidly induced, generally withinminutes of contact between the receptor and the effector protein orligand. The induction of immediate early gene transcription does notrequire the synthesis of new regulatory proteins. In addition to rapidresponsiveness to ligand binding, characteristics of preferred genesuseful to make reporter constructs include: low or undetectableexpression in quiescent cells; induction that is transient andindependent of new protein synthesis; subsequent shut-off oftranscription requires new protein synthesis; and mRNAs transcribed fromthese genes have a short half-life. It is preferred, but not necessarythat a transcriptional control element have all of these properties forit to be useful.

An example of a gene that is responsive to a number of different stimuliis the c-fos proto-oncogene. The c-fos gene is activated in aprotein-synthesis-independent manner by growth factors, hormones,differentiation-specific agents, stress, and other known inducers ofcell surface proteins. The induction of c-fos expression is extremelyrapid, often occurring within minutes of receptor stimulation. Thischaracteristic makes the c-fos regulatory regions particularlyattractive for use as a reporter of receptor activation.

The c-fos regulatory elements include (see, Verma et al., 1987, Cell 51:513-514): a TATA box that is required for transcription initiation; twoupstream elements for basal transcription, and an enhancer, whichincludes an element with dyad symmetry and which is required forinduction by TPA, serum, EGF, and PMA.

The 20 bp c-fos transcriptional enhancer element located between −317and −298 bp upstream from the c-fos mRNA cap site, is essential forserum induction in serum starved NIH 3T3 cells. One of the two upstreamelements is located at −63 to −57 and it resembles the consensussequence for cAMP regulation.

The transcription factor CREB (cyclic AMP responsive element bindingprotein) is, as the name implies, responsive to levels of intracellularcAMP. Therefore, the activation of a receptor that signals viamodulation of cAMP levels can be monitored by measuring either thebinding of the transcription factor, or the expression of a reportergene linked to a CREB-binding element (termed the CRE, or cAMP responseelement). The DNA sequence of the CRE is TGACGTCA (SEQ ID NO: 75).Reporter constructs responsive to CREB binding activity are described inU.S. Pat. No. 5,919,649.

Other promoters and transcriptional control elements, in addition to thec-fos elements and CREB-responsive constructs, include the vasoactiveintestinal peptide (VIP) gene promoter (cAMP responsive; Fink et al.,1988, Proc. Natl. Acad. Sci. 85:6662-6666); the somatostatin genepromoter (cAMP responsive; Montminy et al., 1986, Proc. Natl. Acad. Sci.8.3:6682-6686); the proenkephalin promoter (responsive to cAMP,nicotinic agonists, and phorbol esters; Comb et al., 1986, Nature323:353-356); the phosphoenolpyruvate carboxy-kinase (PEPCK) genepromoter (cAMP responsive; Short et al., 1986, J. Biol. Chem.261:9721-9726).

Additional examples of transcriptional control elements that areresponsive to changes in GPCR activity include, but are not limited tothose responsive to the AP-1 transcription factor and those responsiveto NF-κB activity. The consensus AP-1 binding site is the palindromeTGA(C/G)TCA (Lee et al., 1987, Nature 325: 368-372; Lee et al., 1987,Cell 49: 741-752). The AP-1 site is also responsible for mediatinginduction by tumor promoters such as the phorbol ester12-O-tetradecanoylphorbol-β-acetate (TPA), and are therefore sometimesalso referred to as a TRE, for TPA-response element. AP-1 activatesnumerous genes that are involved in the early response of cells togrowth stimuli. Examples of AP-1-responsive genes include, but are notlimited to the genes for Fos and Jun (which proteins themselves make upAP-1 activity), Fos-related antigens (Fra) 1 and 2, IκBα, ornithinedecarboxylase, and annexins I and II.

The NF-κB binding element has the consensus sequence GGGGACTTTCC (SEQ IDNO: 81). A large number of genes have been identified as NF-κBresponsive, and their control elements can be linked to a reporter geneto monitor GPCR activity. A small sample of the genes responsive toNF-κB includes those encoding IL-1β (Hiscott et al., 1993, Mol. Cell.Biol. 13: 6231-6240), TNF-α (Shakhov et al., 1990, J. Exp. Med. 171:35-47), CCR5 (Liu et al., 1998, AIDS Res. Hum. Retroviruses 14:1509-1519), P-selectin (Pan & McEver, 1995, J. Biol. Chem. 270:23077-23083), Fas ligand (Matsui et al., 1998, J. Immunol. 161:3469-3473), GM-CSF (Schreck & Baeuerle, 1990, Mol. Cell. Biol. 10:1281-1286) and IκBα (Haskill et al., 1991, Cell 65: 1281-1289). Each ofthese references is incorporated herein by reference. Vectors encodingNF-κB-responsive reporters are also known in the art or can be readilymade by one of skill in the art using, for example, synthetic NF-κBelements and a minimal promoter, or using the NF-κB-responsive sequencesof a gene known to be subject to NF-κB regulation. Further, NF-κBresponsive reporter constructs are commercially available from, forexample, CLONTECH.

A given promoter construct should be tested by exposingChemerinR-expressing cells, transfected with the construct, to aChemerin polypeptide. An increase of at least two-fold in the expressionof reporter in response to Chemerin polypeptide indicates that thereporter is an indicator of ChemerinR activity.

In order to assay ChemerinR activity with a Chemerin-responsivetranscriptional reporter construct, cells that stably express aChemerinR polypeptide are stably transfected with the reporterconstruct. To screen for agonists, the cells are left untreated, exposedto candidate modulators, or exposed to a Chemerin polypeptide, andexpression of the reporter is measured. The Chemerin-treated culturesserve as a standard for the level of transcription induced by a knownagonist. An increase of at least 50% in reporter expression in thepresence of a candidate modulator indicates that the candidate is amodulator of ChemerinR activity. An agonist will induce at least asmuch, and preferably the same amount or more, reporter expression thanthe Chemerin polypeptide. This approach can also be used to screen forinverse agonists where cells express a ChemerinR polypeptide at levelssuch that there is an elevated basal activity of the reporter in theabsence of Chemerin or another agonist. A decrease in reporter activityof 10% or more in the presence of a candidate modulator, relative to itsabsence, indicates that the compound is an inverse agonist.

To screen for antagonists, the cells expressing ChemerinR and carryingthe reporter construct are exposed to a Chemerin polypeptide (or anotheragonist) in the presence and absence of candidate modulator. A decreaseof 10% or more in reporter expression in the presence of candidatemodulator, relative to the absence of the candidate modulator, indicatesthat the candidate is a modulator of ChemerinR activity.

Controls for transcription assays include cells not expressing ChemerinRbut carrying the reporter construct, as well as cells with apromoterless reporter construct. Compounds that are identified asmodulators of ChemerinR-regulated transcription should also be analyzedto determine whether they affect transcription driven by otherregulatory sequences and by other receptors, in order to determine thespecificity and spectrum of their activity.

The transcriptional reporter assay, and most cell-based assays, are wellsuited for screening expression libraries for proteins for those thatmodulate ChemerinR activity. The libraries can be, for example, cDNAlibraries from natural sources, e.g., plants, animals, bacteria, etc.,or they can be libraries expressing randomly or systematically mutatedvariants of one or more polypeptides. Genomic libraries in viral vectorscan also be used to express the mRNA content of one cell or tissue, inthe different libraries used for screening of ChemerinR.

Any of the assays of receptor activity, including the GTP-binding,GTPase, adenylate cyclase, cAMP, phospholipid-breakdown, diacylglyceorl,inositol triphosphate, PKC, kinase and transcriptional reporter assays,can be used to determine the presence of an agent in a sample, e.g., atissue sample, that affects the activity of the ChemerinR receptormolecule. To do so, ChemerinR polypeptide is assayed for activity in thepresence and absence of the sample or an extract of the sample. Anincrease in ChemerinR activity in the presence of the sample or extractrelative to the absence of the sample indicates that the sample containsan agonist of the receptor activity. A decrease in receptor activity inthe presence of Chemerin or another agonist and the sample, relative toreceptor activity in the presence of Chemerin polypeptide alone,indicates that the sample contains an antagonist of ChemerinR activity.If desired, samples can then be fractionated and further tested toisolate or purify the agonist or antagonist. The amount of increase ordecrease in measured activity necessary for a sample to be said tocontain a modulator depends upon the type of assay used. Generally, a10% or greater change (increase or decrease) relative to an assayperformed in the absence of a sample indicates the presence of amodulator in the sample. One exception is the transcriptional reporterassay, in which at least a two-fold increase or 10% decrease in signalis necessary for a sample to be said to contain a modulator. It ispreferred that an agonist stimulates at least 50%, and preferably 75% or100% or more, e.g., 2-fold, 5-fold, 10-fold or greater receptoractivation than wild-type Chemerin.

Other functional assays include, for example, microphysiometer orbiosensor assays (see Hafner, 2000, Biosens. Bioelectron. 15: 149-158,incorporated herein by reference).

II. Diagnostic Assays Based upon the Interaction of ChemerinR andChemerin:

Signaling through GPCRs is instrumental in the pathology of a largenumber of diseases and disorders. ChemerinR, which is expressed in cellsof the lymphocyte lineages and which has been shown to act as aco-receptor for immunodeficiency viruses can have a role in immuneprocesses, disorders or diseases. The ChemerinR expression pattern alsoincludes bone and cartilage, indicating that this receptor can play arole in diseases, disorders or processes (e.g., fracture healing)affecting these tissues. Expression in adult parathyroid suggestspossible importance in phosphocalic metabolism.

Because of its expression in cells of the lymphocyte lineages, ChemerinRcan be involved in the body's response to viral infections or indiseases induced by various viruses, including HIV types I and II, orbacteria. The expression pattern of ChemerinR and the knowledge withrespect to disorders generally mediated by GPCRs suggests that ChemerinRcan be involved in disturbances of cell migration, cancer, developmentof tumors and tumor metastasis, inflammatory and neo-plastic processes,wound and bone healing and dysfunction of regulatory growth functions,diabetes, obesity, anorexia, bulimia, acute heart failure, hypotension,hypertension, urinary retention, osteoporosis, angina pectoris,myocardial infarction, restenosis, atherosclerosis, diseasescharacterised by excessive smooth muscle cell proliferation, aneurysms,diseases characterised by loss of smooth muscle cells or reduced smoothmuscle cell proliferation, stroke, ischemia, ulcers, allergies, benignprostatic hypertrophy, migraine, vomiting, psychotic and neurologicaldisorders, including anxiety, schizophrenia, manic depression,depression, delirium, dementia and severe mental retardation,degenerative diseases, neurodegenerative diseases such as Alzheimer'sdisease or Parkinson's disease, and dyskinasias, such as Huntington'sdisease or Gilles de la Tourett's syndrome and other related diseases.

The interaction of ChemerinR with Chemerin can be used as the basis ofassays for the diagnosis or monitoring of diseases, disorders orprocesses involving ChemerinR signaling. Diagnostic assays forChemerinR-related diseases or disorders can have several differentforms. First, diagnostic assays can measure the amount of ChemerinRand/or Chemerin polypeptide, genes or mRNA in a sample of tissue. Assaysthat measure the amount of mRNA encoding either or both of thesepolypeptides also fit in this category. Second, assays can evaluate thequalities of the receptor or the ligand. For example, assays thatdetermine whether an individual expresses a mutant or variant form ofeither ChemerinR or Chemerin, or both, can be used diagnostically.Third, assays that measure one or more activities of ChemerinRpolypeptide can be used diagnostically.

A. Assays that Measure the Amount of ChemerinR or Chemerin

ChemerinR and Chemerin levels can be measured and compared to standardsin order to determine whether an abnormal level of the receptor or itsligand is present in a sample, either of which indicate probabledysregulation of ChemerinR signaling. Polypeptide levels are measured,for example, by immunohistochemistry using antibodies specific for thepolypeptide. A sample isolated from an individual suspected of sufferingfrom a disease or disorder characterized by ChemerinR activity iscontacted with an antibody for ChemerinR or Chemerin, and binding of theantibody is measured as known in the art (e.g., by measurement of theactivity of an enzyme conjugated to a secondary antibody).

Another approach to the measurement of ChemerinR and/or Chemerinpolypeptide levels uses flow cytometry analysis of cells from anaffected tissue. Methods of flow cytometry, including the fluorescentlabeling of antibodies specific for ChemerinR or Chemerin, are wellknown in the art. Other approaches include radioimmunoassay or ELISA.Methods for each of these are also well known in the art.

The amount of binding detected is compared to the binding in a sample ofsimilar tissue from a healthy individual, or from a site on the affectedindividual that is not so affected. An increase of 10% or more relativeto the standard is diagnostic for a disease or disorder characterized byChemerinR dysregulation.

ChemerinR and Chemerin expression can also be measured by determiningthe amount of mRNA encoding either or both of the polypeptides in asample of tissue. mRNA can be quantitated by quantitative orsemi-quantitative PCR. Methods of “quantitative” amplification are wellknown to those of skill in the art, and primer sequences for theamplification of both ChemerinR and Chemerin are disclosed herein. Acommon method of quantitative PCR involves simultaneously co-amplifyinga known quantity of a control sequence using the same primers. Thisprovides an internal standard that can be used to calibrate the PCRreaction. Detailed protocols for quantitative PCR are provided in PCRProtocols, A Guide to Methods and Applications, Innis et al., AcademicPress, Inc. N.Y., (1990), which is incorporated herein by reference. Anincrease of 10% or more in the amount of mRNA encoding ChemerinR orChemerin in a sample, relative to the amount expressed in a sample oflike tissue from a healthy individual or in a sample of tissue from anunaffected location in an affected individual is diagnostic for adisease or disorder characterized by dysregulation of ChemerinRsignaling.

B. Qualitative Assays

Assays that evaluate whether or not the ChemerinR polypeptide or themRNA encoding it are wild-type or not can be used diagnostically. Inorder to diagnose a disease or disorder characterized by ChemerinR orChemerin dysregulation in this manner, RNA isolated from a sample isused as a template for PCR amplification of Chemerin and/or ChemerinR.The amplified sequences are then either directly sequenced usingstandard methods, or are first cloned into a vector, followed bysequencing. A difference in the sequence that changes one or moreencoded amino acids relative to the sequence of wild-type ChemerinR orChemerin can be diagnostic of a disease or disorder characterized bydysregulation of ChemerinR signaling. It can be useful, when a change incoding sequence is identified in a sample, to express the variantreceptor or ligand and compare its activity to that of wild typeChemerinR or Chemerin. Among other benefits, this approach can providenovel mutants, including constitutively active and null mutants.

In addition to standard sequencing methods, amplified sequences can beassayed for the presence of specific mutations using, for example,hybridization of molecular beacons that discriminate between wild-typeand variant sequences. Hybridization assays that discriminate on thebasis of changes as small as one nucleotide are well known in the art.Alternatively, any of a number of “minisequencing” assays can beperformed, including, those described, for example, in U.S. Pat. Nos.5,888,819, 6,004,744 and 6,013,431 (incorporated herein by reference).These assays and others known in the art can determine the presence, ina given sample, of a nucleic acid with a known polymorphism.

If desired, array or microarray-based methods can be used to analyze theexpression or the presence of mutation, in ChemerinR or Chemerinsequences. Array-based methods for minisequencing and for quantitationof nucleic acid expression are well known in the art.

C. Functional Assays.

Diagnosis of a disease or disorder characterized by the dysregulation ofChemerinR signaling can also be performed using functional assays. To doso, cell membranes or cell extracts prepared from a tissue sample areused in an assay of ChemerinR activity as described herein (e.g., ligandbinding assays, the GTP-binding assay, GTPase assay, adenylate cyclaseassay, cAMP assay, phospholipid breakdown, diacyl glycerol or inositoltriphosphate assays, PKC activation assay, or kinase assay). Theactivity detected is compared to that in a standard sample taken from ahealthy individual or from an unaffected site on the affectedindividual. As an alternative, a sample or extract of a sample can beapplied to cells expressing ChemerinR, followed by measurement ofChemerinR signaling activity relative to a standard sample. A differenceof 10% or more in the activity measured in any of these assays, relativeto the activity of the standard, is diagnostic for a disease or disordercharacterized by dysregulation of ChemerinR signaling.

Modulation of ChemerinR Activity in a Cell According to the Invention

The discovery of Chemerin as a ligand of ChemerinR provides methods ofmodulating the activity of a ChemerinR polypeptide in a cell. ChemerinRactivity is modulated in a cell by delivering to that cell an agent thatmodulates the function of a ChemerinR polypeptide. This modulation canbe performed in cultured cells as part of an assay for theidentification of additional modulating agents, or, for example, in ananimal, including a human. Agents include Chemerin polypeptides asdefined herein, as well as additional modulators identified using thescreening methods described herein.

An agent can be delivered to a cell by adding it to culture medium. Theamount to deliver will vary with the identity of the agent and with thepurpose for which it is delivered. For example, in a culture assay toidentify antagonists of ChemerinR activity, one will preferably add anamount of Chemerin polypeptide that half-maximally activates thereceptors (e.g., approximately EC₅₀), preferably without exceeding thedose required for receptor saturation. This dose can be determined bytitrating the amount of Chemerin polypeptide to determine the point atwhich further addition of Chemerin has no additional effect on ChemerinRactivity.

When a modulator of ChemerinR activity is administered to an animal forthe treatment of a disease or disorder, the amount administered can beadjusted by one of skill in the art on the basis of the desired outcome.Successful treatment is achieved when one or more measurable aspects ofthe pathology (e.g., tumor cell growth, accumulation of inflammatorycells) is changed by at least 10% relative to the value for that aspectprior to treatment.

Candidate Modulators Useful According to the Invention

Candidate modulators can be screened from large libraries of syntheticor natural compounds. Numerous means are currently used for random anddirected synthesis of saccharide, peptide, lipid, carbohydrate, andnucleic acid based compounds. Synthetic compound libraries arecommercially available from a number of companies including, forexample, Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex(Princeton, N.J.), Brandon Associates (Merrimack, N.H.), and Microsource(New Milford, Conn.). A rare chemical library is available from Aldrich(Milwaukee, Wis.). Combinatorial libraries of small organic moleculesare available and can be prepared. Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant and animal extractsare available from e.g., Pan Laboratories (Bothell, Wash.) or MycoSearch(NC), or are readily produceable by methods well known in the art.Additionally, natural and synthetically produced libraries and compoundsare readily modified through conventional chemical, physical, andbiochemical means.

As noted previously herein, candidate modulators can also be variants ofknown polypeptides (e.g., Chemerin, antibodies) or nucleic acids (e.g.,aptamers) encoded in a nucleic acid library. Cells (e.g., bacteria,yeast or higher eukaryotic cells) transformed with the library can begrown and prepared as extracts, which are then applied in ChemerinRbinding assays or functional assays of ChemerinR activity.

III. Antibodies Useful According to the Invention

The invention provides for antibodies to ChemerinR and Chemerin.Antibodies of the invention include, but are not limited to, polyclonal,monoclonal, multispecific, human, humanized or chimeric antibodies,single-chain antibodies, Fab fragments, F(ab′) fragments, etc. Theantibodies of the invention can be any type (e.g., IgG, IgE, IgM, IgD,IgA, and IgY), class (e.g., IgG1-4, IgA1-2), or subclass ofimmunoglobulin molecule. In a preferred embodiment, the antibody is anIgG isotype. In another preferred embodiment, the antibody is an IgG1isotype. In another preferred embodiment, the antibody is an IgG2isotype. In another preferred embodiment, the antibody is an IgG4isotype.

The antibodies of the invention may bind specifically to a polypeptideor polypeptide fragment or variant of Chemerin. Preferably, theantibodies of the invention bind specifically to the full-lengthChemerin polypeptide. Also preferably, the antibodies of the inventionbind specifically to the 157 amino acid truncated Preprochemerinpolypeptide (SEQ ID NO: 73). Also preferably, the antibodies of theinvention bind specifically to the 19 amino acid Chemerin polypeptide(SEQ ID NO: 53). Also preferbly, the antibodies of the invention bindspecifically to the 9 amino acid Chemerin polypeptide (SEQ ID NO: 59).Also preferably, the antibodies of the invention bind specifically tothe Chemerin fragment FSKALPRS (SEQ ID NO: 89).

The antibodies of the invention may act as agonists or antagonists ofthe polypeptides of the invention. For example, the antibodies of theinvention disrupt the Chemerin/ChemerinR interactions. The inventionalso features the antibodies that do not disrupt the Chemerin/ChemerinRinteractions but disrupt the ChemerinR activation.

The antibodies of the invention may be used, for example, but notlimited to, to purify, detect, and target the polypeptides of theinvention, including both in vitro and in vivo diagnostic andtherapeutic methods. For example, the antibodies of the invention can beused in immunoassays for qualitatively and quantitatively measuringlevels of the polypeptides of the present invention in biologicalsamples (Antibodies: A Laboratory Manual, Ed. by Harlow and Lane (ColdSpring Harbor Press: 1988)). The antibodies of the invention may be usedeither alone or in combination with other compositions. The antibodiesmay further be recombinantly fused to a heterologous polypeptide at theN- or C-terminus or chemically conjugated (including covalently andnon-covalently conjugations) to polypeptides or other compositions. Theantibodies of the invention may also be modified by the covalentattachment of any type of molecule to the antibodies, including byglycosylation, acetylation, pegylation, phosphylation, phosphorylation,amidation, derivatization by known protecting/blocking groups,proteolytic cleavage, linkage to a cellular ligand or other protein,etc.

Antibodies can be made using standard protocols known in the art (See,for example, Antibodies: A Laboratory Manual, Ed. by Harlow and Lane(Cold Spring Harbor Press: 1988)). A mammal, such as a mouse, hamster,or rabbit can be immunized with an immunogenic form of the peptide(e.g., a ChemerinR or Chemerin polypeptide or an antigenic fragmentwhich is capable of eliciting an antibody response, or a fusion proteinas described herein above). Immunogens for raising antibodies areprepared by mixing the polypeptides (e.g., isolated recombinantpolypeptides or synthetic peptides) with adjuvant. Alternatively,ChemerinR or Chemerin polypeptides or peptides are made as fusionproteins to larger immunogenic proteins. Polypeptides can also becovalently linked to other larger immunogenic proteins, such as keyholelimpet hemocyanin. Alternatively, plasmid or viral vectors encodingChemerinR or Chemerin, or a fragment of these proteins, can be used toexpress the polypeptides and generate an immune response in an animal asdescribed in Costagliola et al., 2000, J. Clin. Invest. 105:803-811,which is incorporated herein by reference. In order to raise antibodies,immunogens are typically administered intradermally, subcutaneously, orintramuscularly to experimental animals such as rabbits, sheep, andmice. In addition to the antibodies discussed above, geneticallyengineered antibody derivatives can be made, such as single chainantibodies.

The progress of immunization can be monitored by detection of antibodytiters in plasma or serum. Standard ELISA, flow cytometry or otherimmunoassays can also be used with the immunogen as antigen to assessthe levels of antibodies. Antibody preparations can be simply serum froman immunized animal, or if desired, polyclonal antibodies can beisolated from the serum by, for example, affinity chromatography usingimmobilized immunogen.

To produce monoclonal antibodies, antibody-producing splenocytes can beharvested from an immunized animal and fused by standard somatic cellfusion procedures with immortalizing cells such as myeloma cells toyield hybridoma cells. Such techniques are well known in the art, andinclude, for example, the hybridoma technique (originally developed byKohler and Milstein, (1975) Nature, 256: 495-497), the human B cellhybridoma technique (Kozbar et al., (1983) Immunology Today, 4:72), andthe EBV-hybridoma technique to produce human monoclonal antibodies (Coleet al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc. pp. 77-96). Hybridoma cells can be screened immunochemically forproduction of antibodies specifically reactive with a Chemerin orChemerinR peptide or polypeptide, and monoclonal antibodies isolatedfrom the media of a culture comprising such hybridoma cells.

Antibody fragments of the invention may be generated by knowntechniques. For example, Fab and F(ab′)2 fragments of the invention maybe produced by proteolytic cleavage of immunoglobulin molecules, usingenzymes such as papain (to produce Fab fragments) or pepsin (to produceF(ab′)2 fragments).

IV. Therapeutic Approaches Based on the Interaction of Chemerin andChemerinR Composition or Therapeutic Composition and AdministrationThereof

The invention provides composition or therapeutic compositions thatcontain a Chemerin polypeptide or a Chemerin nucleic acid sequence asdescribed above. The therapeutic compositions comprise a therapeuticallyeffective amount of a compound including a Chemerin, and apharmaceutically acceptable carrier. In a preferred embodiment, thecomposition is formulated in accordance with routine procedures as apharmaceutical composition adapted for intravenous administration tohuman beings.

In another preferred embodiment, the composition of the invention can beformulated as neutral or salt forms. Pharmaceutically acceptable saltsinclude those formed with anions such as those derived fromhydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., andthose formed with cations such as those derived from sodium, potassium,ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine,2-ethylamino ethanol, histidine, procaine, etc.

Generally, a composition will be administered in a single dosage in therange of 100 μg-100 mg/kg body weight, preferably in the range of 1μg-100 μg/kg body weight. This dosage may be repeated daily, weekly,monthly, yearly, or as considered appropriate by the treating physician.Alternatively, the therapeutically effective amount of the compositionof the invention can be determined by standard clinical techniques. Inaddition, in vitro assays may optionally be employed to help identifyoptimal dosage ranges. The precise dose to be employed in theformulation will also depend on the route of administration, and theseriousness of the disease or disorder, and should be decided accordingto the judgment of the practitioner and each patient's circumstances.Effective doses may be extrapolated from dose-response curves derivedfrom in vitro or animal model test systems.

The invention also provides methods of treatment and inhibition for adisease or disorder by administration to a subject of an effectiveamount of a composition or therapeutic composition of the invention,preferably a nucleic acid or a polypeptide Chemerin molecule. In oneaspect, the composition is substantially free from substances that limiteffect or produce undesired side-effects of Chemerin. The subject can beany animal, and is preferably a mammal, and preferably a human.

Various delivery systems known in the art can be used to administer acomposition of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe composition of the invention, receptor-mediated endocytosis, etc,which are incorporated by reference herein. Methods of introductioninclude but are not limited to intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The compositions of the invention may be administered byany convenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents. Administration can besystemic or local. In addition, it may be desirable to introduce thecompositions or the therapeutic compositions of the invention into thecentral nervous system by any suitable route, including intraventricularand intrathecal injection; intraventricular injection may be facilitatedby an intraventricular catheter, for example, attached to a reservoir,such as an Ommaya reservoir. Pulmonary administration can also beemployed, e.g., by use of an inhaler or nebulizer, and formulation withan aerosolizing agent.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention.

Various diseases or disorders can be treated with the compositions ortherapeutic compositions of the invention. They include, but are notlimited to, neoplasms located in the: colon, abdomen, bone, breast,digestive system, liver, pancreas, peritoneum, endocrine glands(adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid),eye, head and neck, nervous (central and peripheral), lymphatic system,pelvic, skin, soft tissue, spleen, thoracic, and urogenital, as well ashypergammaglobulinemia, lymphoproliferative diseases, disorders, and/orconditions, paraproteinemias, purpura, sarcoidosis, Sezary Syndrome,Waldenstron's Macroglobulinemia, Gaucher's Disease, histiocytosis, andany other hyperproliferative disease.

Transgenic Animals Useful According to the Invention

Transgenic animals expressing ChemerinR or Chemerin or variants thereofare useful to study the signaling through ChemerinR, as well as for thestudy of drugs or agents that modulate the activity of ChemerinR. Atransgenic animal is a non-human animal containing at least one foreigngene, called a transgene, which is part of its genetic material.Preferably, the transgene is contained in the animal's germ line suchthat it can be transmitted to the animal's offspring. A number oftechniques may be used to introduce the transgene into an animal'sgenetic material, including, but not limited to, microinjection of thetransgene into pronuclei of fertilized eggs and manipulation ofembryonic stem cells (U.S. Pat. No. 4,873,191 by Wagner and Hoppe;Palmiter and Brinster, 1986, Ann. Rev. Genet., 20:465-499; French PatentApplication 2593827 published Aug. 7, 1987, all of which areincorporated herein by reference). Transgenic animals can carry thetransgene in all their cells or can be genetically mosaic.

According to the method of conventional transgenesis, additional copiesof normal or modified genes are injected into the male pronucleus of thezygote and become integrated into the genomic DNA of the recipientmouse. The transgene is transmitted in a Mendelian manner in establishedtransgenic strains. Transgenes can be constitutively expressed or can betissue specific or even responsive to an exogenous drug, e.g.,Tetracycline. A transgenic animal expressing one transgene can becrossed to a second transgenic animal expressing a second transgene suchthat their offspring will carry and express both transgenes.

Knock-Out Animals

Animals bearing a homozygous deletion in the chromosomal sequencesencoding either ChemerinR or Chemerin or variants can be used to studythe function of the receptor and ligand. Of particular interest iswhether a Chemerin knockout has a distinct phenotype, which may point towhether Chemerin is the only ligand that binds ChemerinR or if it is amember of a family. Of further particular interest is the identificationof identification of ChemerinR/Chemerin in specific physiological and/orpathological processes.

i. Standard Knock Out Animals

Knock out animals are produced by the method of creating gene deletionswith homologous recombination. This technique is based on thedevelopment of embryonic stem (ES) cells that are derived from embryos,are maintained in culture and have the capacity to participate in thedevelopment of every tissue in the animals when introduced into a hostblastocyst. A knock out animal is produced by directing homologousrecombination to a specific target gene in the ES cells, therebyproducing a null allele of the gene. The technology for making knock-outanimals is well described (see, for example, Huszar et al., 1997, Cell,88:131; and Ohki-Hamazaki et al., 1997, Nature, 390:165, both of whichare incorporated herein by reference). One of skill in the art cangenerate a homozygous ChemerinR or Chemerin knock-out animal (e.g., amouse) using the sequences for ChemerinR and Chemerin (disclosed hereinand known in the art) to make the gene targeting construct.

ii. Tissue Specific Knock Out

The method of targeted homologous recombination has been improved by thedevelopment of a system for site-specific recombination based on thebacteriophage P1 site specific recombinase Cre. The Cre-loxPsite-specific DNA recombinase from bacteriophage P1 is used intransgenic mouse assays in order to create gene knockouts restricted todefined tissues or developmental stages. Regionally restricted geneticdeletion, as opposed to global gene knockout, has the advantage that aphenotype can be attributed to a particular cell/tissue (Marth, 1996,Clin. Invest. 97: 1999). In the Cre-loxP system one transgenic mousestrain is engineered such that loxP sites flank one or more exons of thegene of interest. Homozygotes for this so called ‘floxed gene’ arecrossed with a second transgenic mouse that expresses the Cre gene undercontrol of a cell/tissue type transcriptional promoter. Cre protein thenexcises DNA between loxP recognition sequences and effectively removestarget gene function (Sauer, 1998, Methods, 14:381). There are now manyin vivo examples of this method, including, for instance, the inducibleinactivation of mammary tissue specific genes (Wagner et al., 1997,Nucleic Acids Res., 25:4323). One of skill in the art can thereforegenerate a tissue-specific knock-out animal in which ChemerinR orChemerin is homozygously eliminated in a chosen tissue or cell type.

Kits Useful According to the Invention

The invention provides for kits useful for screening for modulators ofChemerinR activity, as well as kits useful for diagnosis of diseases ordisorders characterized by dysregulation of ChemerinR signaling. Kitsuseful according to the invention can include an isolated ChemerinRpolypeptide (including a membrane- or cell-associated ChemerinRpolypeptide, e.g., on isolated membranes, cells expressing ChemerinR,or, on an SPR chip) and an isolated Chemerin polypeptide. A kit can alsocomprise an antibody specific for ChemerinR and/or an antibody forChemerin. Alternatively, or in addition, a kit can contain cellstransformed to express a ChemerinR polypeptide and/or cells transformedto express a Chemerin polypeptide. In a further embodiment, a kitaccording to the invention can contain a polynucleotide encoding aChemerinR polypeptide and/or a polynucleotide encoding a Chemerinpolypeptide. In a still further embodiment, a kit according to theinvention may comprise the specific primers useful for amplification ofChemerinR or Chemerin as described below. All kits according to theinvention will comprise the stated items or combinations of items andpackaging materials therefor. Kits will also include instructions foruse.

Expression Vectors

The present invention also relates to vectors containing the Chemerinand host cells, as well as the production of the Chemerin polypeptide byrecombinant techniques. The vector may be a phage, plasmid, viral, orretroviral vector. The Chemerin polynucleotides may be joined to avector containing a selectable marker propagation in a host. TheChemerin polynucleotide should be operatively linked to an appropriatepromoter, as the phage lambda PL promoter, the E. coli lac, trp, phoAand tac promoters, the SV40 early and late promoters and promoters ofretroviral LTRs. The expression vectors will further contain sites fortranscription initiation, termination, and, in the transcribed region, aribosome binding site for translation. The coding portion of thetranscripts expressed by the constructs will preferably include atranslation initiating codon at the beginning and a termination codon(UAA, UGA or UAG) appropriately positioned at the end of the polypeptideto be translated. The expressing vectors will also include one or morepromoters. Suitable promoters which may be employed include, but are notlimited to, retroviral LTR, the SV40 promoter, adenoviral promoters;heterologous promoters, such as the cytomegalovirus (CMV) promoter; therespiratory syncytial virus (RSV) promoter; inducible promoters, such asthe MMT promoter, the metallothionein promoter; heat shock promoters;the albumin promoter; the ApoAI promoter; human globin promoters; viralthymidine kinase promoters, such as the Herpes Simplex thymidine kinasepromoter; retroviral LTRs (including the modified retroviral LTRshereinabove described).; the .beta.-actin promoter; and human growthhormone promoters. The promoter also may be the native promoter whichcontrols the genes encoding the polypeptides.

As indicated, the expression vectors will preferably include at leastone selectable marker. Such markers include dihydrofolate reductase,G418, glutamine synthase or neomycin resistance for eukaryotic cellculture and tetracycline, kanamycin or ampicillin resistance genes forculturing in E. coli and other bacteria. Representative examples ofappropriate hosts include, but are not limited to, bacterial cells, suchas E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells,such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris(ATCC Accession No. 201178)); insect cells such as Drosophila S2 andSpodoptera Sf9 cells; animal cells such as CHO, NSO, COS, 293, and Bowesmelanoma cells; and plant cells. Appropriate culture mediums andconditions for the above-described host cells are known in the art.

Gene Transfer Methods

Gene therapy has been studied and used for treating various types ofdiseases. Generally, gene therapy comprises delivering a gene ofinterest to cells affected with diseases for correction of abnormalconditions. The invention provides for gene transfer methods of theChemerin gene for treatment of diseases including tumors/cancers such ascancers in lung, prostate, oesophagus, Pharynx, Colon-rectum,liver-bilary tract, stomach, larynx, pancreas, bladder, breast,colon-rectum, ovary, stomach, womb-leasing, pancreas, lung, liver,lymphoma, leukemia. Gene transfer of the Chemerin gene in accordancewith the present invention can be accomplished through many means,including by both viral vectors and by non-viral methods.

The non-viral gene transfer methods include plasmid DNA expressionvectors, liposomes, receptor-mediated endocytosis, and particle-mediated(gene gun) methods etc. All these methods are well known in the art andare incorporated by reference herein.

The viral gene transfer methods include retrovirus (includinglentivirus), adenovirus, adeno-associated virus, herpes simplex virus,vaccinia, fowlpox, canarypox virus, Sindbis virus etc, which are wellknown in the art. In one embodiment, the gene transfer relates torecombinant retrovirus vectors such as the virus based on Mouse MoloneyLeukemia virus, the chimeric Moloney-Human lentiviral (HIV) vector etc.

In another embodiment, the gene transfer relates to human adenoviruses.The human adenovirus is a 36 kb double-stranded DNA virus containinggenes that express more than 50 gene products throughout its life cycle.By eliminating the E1 region of the vector, the virus lacks ability toself-replicate and space is made for placing therapeutic expressionsequences. The adenovirus vectors have been shown to be especiallyefficient at transferring genes into most tissues after in vivoadministration. In another particular embodiment, the adenovirus vectorcan be modified to exhibit tissue-specific, tumor-selective expression(Doronin, K et al. (2001) J. Virology, 75:3314-3324). In one example,the adenovirus promoter E1A region is deleted and replaced with amodified promoter for α-fectoprotein (AFP). The expression of thismodified adenovirus vector is limited to hepatocellular carcinoma cells(Hallenbeck, P L et al. (1999) Human Gene Ther. 10:1721-1733). Inanother example, the adenovirus E4 promoter region is deleted andreplaced with the promoter for surfactant protein B (SPB). Theexpression of the modified adenovirus is limited to lung carcinoma cells(Doronin, K et al. (2001) J. Virology, 75:3314-3324).

In another embodiment, the gene transfer relates to recombinantadeno-associated virus (AAV) vectors. The AAV vectors contain small,single-stranded DNA genomes and have been shown to transduce brain,skeletal muscle, and liver tissues.

The cells targeted for gene transfer include any cells to which thedelivery of the Chemerin gene is desired. Generally, the cells are thoseaffected with diseases such as but not limited to tumoric cells. Variousmammalian cell lines can also be employed for gene transfer, examplesincludes, but not limited to, COS-7 lines of monkey kidney fibroblasts,described by Gluzman, Cell 23:175 (1981), and other cell lines capableof expressing a compatible vector, for example, the C127, 3T3, CHO, HeLaand BHK cell lines. In particular, the cells are cell lines derived fromtissues affected by diseases, such as cancer cell lines.

Ex Vivo Therapeutic Approaches Based On The Interaction of Chemerin andChemerinR

The ex vivo gene therapy involves removing cells from the blood ortissues of a subject, genetically modifying in vitro, and subsequentlytransplanting back into the same recipient. In one embodiment, a nucleicacid sequence is introduced into a cell prior to administration in vivoof the resulting recombination cell. Such introduction can be carriedout by any method known in the art, including but not limited totransfection, electroporation, microinjection, infection with a viral orbacteriophage vector containing the nucleic acid sequence, cell fusion,chromosome-mediated gene transfer, microcell-mediated gene transfer,shperoplast fusion, etc, all are known in the art. The gene transfermethods should provide for stable transfer of the nucleic acid sequenceto the cell, so that the nucleic acid sequence is expressible in thecell and preferably heritable and expressible by its cell progeny. Theresulting recombinant blood cells are preferably administeredintravenously. The amount of cells envisioned for use depends on thedesired effect, patient state, etc., and can be determined by oneskilled in the art. Cells into which a nucleic acid can be introducedfor purposes of gene therapy encompass any desired, available cell type,and include but are not limited to epithelial cells, endothelial cells,keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells suchas T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc. In a preferred embodiment, the nucleic acid sequence encodesa Chemerin polypeptide including the polypeptides ranging from thetruncated to the full-length and the variants of the Chemerinpolypeptide that bind specifically to a ChemerinR polypeptide. In apreferred embodiment, the cell used for ex vivo gene therapy isautologous to the recipient.

In another preferred embodiment, cells used are dendritic cells. Forexample, dendritic cells can be derived from hematopoietic progenitorsor from adherent peripheral blood monocytes. The cultured dendriticcells are then loaded with tumor-associated antigens. Tumor antigenloading can be accomplished by a variety of techniques including (1)pulsing with purified defined peptides or modified tumor lysate, (2)co-culture with apoptotic tumor cells, (3) transfection with RNA, (4)fusion with tumor cells, or (5) gene transfer with viral or non-viralgene transfer systems as described above. The loaded dendritic cells areinjected into a subject for stimulating immune response of the subject.

In another embodiment, cells can be pulsed with different types ofcompositions, preferably proteins or peptides. Such technique is knownto one skilled in the art and is described in Nestle et al. (1998) Nat.Med. 4:328-332. Briefly, cells are transferred into a suitable mediumand incubated in vitro for an appropriate time with the composition. Thecells are then washed and resuspended in a suitable volume of medium forin vivo transfer. In a preferred embodiment, the cells used for peptidepulsing are of the same species as the individual to whom thecomposition should be applied. In a particularly preferred embodiment,the cells are autologous to the recipient. In another particularlypreferred embodiment, the cells are dendritic cells.

Particular examples of ex vivo dendritic cell gene therapy include thosethat have been assessed in melanoma (Nestle et al. (1998) Nat. Med.4:328-332), renal cancer (Kurokawa et al. (2001), Int. J. Cancer,91:749-756), glioma (Yu et al (2001), Cancer Res., 61:842-847), breastand ovarian (Brossart et al. (2000), Blood, 96:3102-3108), prostate(Burch et al. (2000), Clin. Cancer Res., 6:2175-2182), gastrointestinal,colon and lung (Fong et al. (2001) J. Immunl., 166:4254-4259).

In Vivo Gene Therapy

The present invention provides in vivo gene therapy methods. Suchmethods involve the direct administration of nucleic acid or a nucleicacid/protein complex into the individual being treated. For example,successful examples of animal models with in vivo gene therapy can befound in treatment of lung cancer (Zhang and Roth (1994), In Vivo,8(5):755-769) and cutaneous melanoma (Gary et al. (1993), PNAS USA,90:11307-11311), etc.

The nucleic acid or protein is preferably Preprochemerin or ChemerinR(SEQ ID NO: 7), truncated Preprochemerin (SEQ ID NO: 72) or ChemerinR(SEQ ID NO: 1) of the invention. In vivo administration can beaccomplished according to a number of established techniques including,but not limited to, injection of naked nucleic acid, viral infection,transport via liposomes and transport by endocytosis as described above.Suitable viral vectors include, for example, adenovirus,adeno-associated virus and retrovirus vectors etc as described in detailabove.

The Preprochemerin or truncated Preprochemerin polynucleotides in avector can be delivered to the interstitial space of tissues with asubject, including of muscle, skin, brain, lung, liver, spleen, bonemarrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney,gall bladder, stomach, intestine, testis, ovary, uterus, rectum, nervoussystem, eye, gland, and connective tissue.

In one embodiment of the invention, the Preprochemerin polynucleotidesor truncated Preprochemerin/Chemerin or truncated Preprochemerinpolypeptides are complexed in a liposome preparation. Liposomalpreparations for use in the present invention include cationic, anionic,and neutral preparations, all are well known in the art.

In one embodiment, a retroviral vector containing a Preprochemerin ortruncated Preprochemerin RNA sequence is used for in vivo gene therapy.In another embodiment of the invention, an adenovirus-associated virusvector containing a preprochemerin or truncated Preprochemerinpolynucleotides is used. In another embodiment, an adenovirus vectorcontaining a Preprochemerin polypeptide is used.

In one embodiment of the invention, the viral vectors for gene transferare adenovirus vectors whose promoters are modified so that theexpression of the vectors is limited to a specific tumor or a particulartissue. This type of vectors have the advantages of delivering the geneof interest to the targeted location, thus reducing the chance of harmdue to the unspecific delivery of the viral vector to a variety oftissues including the normal cell tissues.

In a particular embodiment of the invention, the in vivo gene therapyincludes administering the gene that encodes a Preprochemerin ortruncated Preprochemerin polypeptide into a subject for stimulatingimmune response of the subject or therapeutic treatment of a disease.Preferably, the gene encoding a Preprochemerin or truncatedPreprochemerin polypeptide is administered by a plasmid vector, or aviral vector, or non-viral methods. Preferably, the gene encoding aPreprochemerin or truncated Preprochemerin polypeptide is administeredby a adenovirus vector whose expression is tissue-specific and/ortumor-selective.

The polynucleotides encoding Preprochemerin or truncated Preprochemerinmay be administered along with other polynucleotides encoding anangiogenic protein. Examples of angiogenic proteins include, but are notlimited to, acidic and basic fibroblast growth factors, VEGF-1, VEGF-2,VEGF-3, epidermal growth factor alpha and beta, platelet-derivedendothelial cell growth factor, platelet-derived growth factor, tumornecrosis factor alpha, hepatocyte growth factor, insulin like growthfactor, colony stimulating factor, macrophage colony stimulating factor,granulocyte/macrophage colony stimulating factor, and nitric oxidesynthase.

Determining an effective amount of substance to be delivered can dependupon a number of factors including, for example, the chemical structureand biological activity of the substance, the age and weight of theanimal, the precise condition requiring treatment and its severity, andthe route of administration. The frequency of treatments depends upon anumber of factors, such as the amount of polynucleotide constructsadministered per dose, as well as the health and history of the subject.The precise amount, number of doses, and timing of doses will bedetermined by the attending physician or veterinarian.

EXAMPLES

In the following examples, all chemicals are obtained from Sigma, unlessstated. The cell culture media are from Gibco BRL and the peptides arefrom Bachem.

Example 1 Cloning of Human ChemerinR Receptor

Human ChemerinR was cloned as described in Samson et al. (1998) (SEQ IDNOS: 1 and 2). As an example of one set of steps one could use to cloneother ChemerinR polypeptides useful according to the invention, themethod is described here. In order to clone the ChemerinR sequence, aclassical cloning procedure was performed on human genomic DNA. A clone,designated HOP 102 (ChemerinR), was amplified from human genomic DNA byusing degenerate oligonucleotides. HOP 102 shared 45-50% identity withFMLP and C5a receptors and somewhat lower similarities with the familyof chemokine receptors (FIG. 5). This partial clone was used as a probeto screen a human genomic library and three overlapping lambda cloneswere isolated. A restriction map of the clones was established and a 1.7kb XbaI fragment was subcloned in pBS SK+ (Stratagene) and sequenced onboth strands. The sequence was found to include the HOP 102 probeentirely, with 100% identity. This novel gene was named ChemerinR(GenBank Accession No. Y14838).

Amplification of coding sequence of ChemerinR resulted in a fragment of1.1 kb. This fragment was subcloned into the pCDNA3 (Invitrogen) vectorand sequenced on both strands (FIGS. 1 and 2).

The mouse and rat ortholog genes are disclosed in FIGS. 3 and 4respectively.

Example 2a Purification of the Natural Ligand of ChemerinR andIdentification of Chemerin

Approximately one liter of a human ascitic fluid from a patient withovarian cancer was prefiltered and then filtered successively through0.45 and 0.22 μm Millex filters (Millipore).

In step 1, the ascite was directly loaded onto a C18 reverse-phasecolumn (10 mm×100 mm POROS 20 R2 beads, Applied Biosystems)pre-equilibrated with 5% CH₃CN/0.1% TFA at a flow-rate of 20 ml/min atroom temperature. A 5-95% gradient of CH₃CN in 0.1% TFA was then appliedwith a slope of 6%/min. 5-milliliter fractions were collected, and 20 μlof each fraction was set aside and assayed for [Ca²⁺] transients inChemerinR-expressing CHO cells.

In step 2, the active fractions (approx. 10 fractions eluting between 25and 40% CH₃CN) were pooled, adjusted at pH 5, filtered through a 20 μmMillex filter (Millipore), diluted 3-fold in acetate buffer at pH 4.8and then applied to a cation-exchange HPLC column (Polycat 9.6 mm×250mm, Vydac) pre-equilibrated with acetate buffer at pH 4.8 and 4° C. A0-1M gradient of NaCl in acetate buffer at pH 4.8 was applied with10%/min at a flow-rate of 4 ml/min. 1-milliliter fractions werecollected and a 25 μl-aliquot from each fraction was used for the [Ca²⁺]assay after desalting on a 10 kDa-cut-off membrane (Ultrafree,Millipore).

In step 3, the active fractions (eluted with approx. 700 mM NaCl) werepooled and desalted onto a 10 kDa-cut-off Ultrafree membrane to approx.10 mM NaCl concentration. The eluates from distinct cation-exchange HPLCruns were pooled and loaded onto a second cation-exchange HPLC column(Polycat 2.1 mm×250 mm, Vydac) pre-equilibrated with acetate buffer atpH 4.8 and 4° C. A 0-1 M gradient of NaCl in acetate buffer at pH 4.8was applied at a flow-rate of 1 ml/min. with a slope of 2%/min.0.5-milliliter fractions were collected and a 20 μl-aliquot from eachfraction was used for intracellular calcium assay after desalting onto a10 kDa-cut-off Ultrafree membrane.

In step 4, the active fractions were pooled, diluted 8-fold withH₂O/0.1% H₃PO₄ and loaded onto an analytical C18 reverse-phase column(4.6 mm×250 mm, Vydac) pre-equilibrated with 5% CH₃CN/0.1% H₃PO₄ at aflow-rate of 1 ml/min at room temperature. A 5-95% gradient of CH₃CN in0.1% H₃PO₄ was applied with a 0.3%/min. gradient between 25 and 40% ofCH₃CN. Individual UV absorption peaks (214 nm) were collected manually,and approx. 5% from each fraction volume was assayed for biologicalactivity.

In step 5, the active peaks (approximatively 28% CH₃CN) were diluted6-fold with H₂O/0.1% TFA and directly loaded onto a second C18reverse-phase column (1 mm×50 mm, Vydac) pre-equilibrated with 5%CH₃CN/0.1% TFA at a flow-rate of 0.1 ml/min. at room temperature. A5-95% gradient of CH₃CN in 0.1% TFA was applied with a 0.3%/min.gradient between 30 and 45% of CH₃CN. The final peak was collectedmanually at 40% CH₃CN and analysed by mass spectrometry. 800 ml ofovarian cancer ascites fluid yielded 50 fmoles of Chemerin.

The active fraction was completely dried in a speed-vac and resuspendedin 10 μl of 0.1M Tris at pH 8.7. After boiling the sample during 15 minat 95° C., the sample was incubated at 37° C. overnight in the presenceof 250 ng of modified trypsin (Promega). The digested sample was thenpurified by solid-phase extraction onto a C18 ZipTip (Millipore). Theeluted sample (1.5 μl in 70% CH₃CN/0.1% TFA) was applied onto a MALDItarget in the presence of 120 mg/ml dihydroxy-benzoic acid matrix andthen analysed on a MALDI-Q-TOF prototype (Micromass). Eight peptideswere predicted to derive from the product of the humantazarotene-induced gene (Tig)-2 (FIG. 20), covering 91 aminoacids out ofthe 143 aminoacid-long sequence of the Tig-2 gene product (after removalof the predicted signal peptide). However, the C-terminal peptide(peptide 8) was not tryptic, lacking the last six amino acids of thepredicted protein. This observation indicated that the active compoundmight result from the proteolytic processing of the encoded precursor(FIGS. 12 and 13).

Example 2b Purification of Human Native Chemerin (FIG. 21)

One liter of ascitic fluid was filtered and loaded (50 ml per run) ontoa reverse-phase column (10×100 mm, Poros 20 R2 beads, AppliedBiosystems). A 5-95% CH₃CN gradient (6%/min) in 0.1% TFA was applied, 5ml fractions were collected and assayed for ChemR23 activation. Activefractions were adjusted to pH 4.8 and applied to a cation-exchange HPLCcolumn (Polycat 9.6×250 mm, Vydac) in the presence of 10% CH₃CN, elutedwith a 0-1 M NaCl gradient (10%/min) in acetate buffer pH 5. Activefractions were desalted (Ultrafree, cut-off: 10 kDa, Millipore), loadedonto a second cation-exchange column (Polycat 2.1×250 mm, Vydac) andeluted with the same buffer (2%/min NaCl gradient). Active fractions(0.5 ml, desalted) were pooled, diluted 8-fold with 0.1% H₃PO₄ andloaded onto a C18 column (4.6×250 mm, Vydac). A 5-95% CH₃CN gradient(0.3%/min) in 0.1% H₃PO₄ was applied and individual UV absorption peaks(214 nm) were collected manually and assayed. The active fractions wereloaded onto a second C18 column (2.1×250 mm, Vydac, 5-95% CH₃CN in 0.1%TFA, 0.3%/min). The peaks were collected manually and analyzed by massspectrometry. The use of human material collected for diagnostic ortherapeutic purposes was approved by the ethical committee of theMedical School of the Université Libre de Bruxelles.

Example 2c Mass Spectrometry Analysis

The active fractions were vacuum dried, resuspended in 10 Ξl of 100 mMTris-HCl pH 8.7, heated for 15 min at 95° C., incubated overnight at 37°C. with 250 ng of trypsin (Promega) and purified by solid-phaseextraction (C18 ZipTip, Millipore). The digested peptides were eluted in1.5 μl of 70% CH₃CN/0.1% TFA onto a metallic MALDI target, dried andthen mixed in 1.5 μl of matrix mix (2 mg/ml 2,5-dihydroxybenzoic acidand 10 mg/ml

-cyano-4-hydroxycinnamic acid, 2 mM fucose, 5 mM ammonium acetate). Forproteic samples excised from SDS/acrylamide gels, the samples wereprocessed as described (14). For determination of the N-terminus of therecombinant protein, the digested peptides were first separated onto aC18 column (1×250 mm, Vydac, 5-95% CH₃CN in 0.1% TFA, 2%/min) and eachHPLC fraction was analyzed separately. Mass spectrometry analysis wasperformed on a Q-TOF Ultima Global mass spectrometer equipped with aMALDI source (Micromass), and calibrated using the monoisotopic massesof tryptic and chymotryptic peptides from bovine serum albumin.Ionization was achieved using a nitrogen laser (337 nm beam, 10 Hz) andacquisitions were performed in a V mode reflectron position.Microsequencing was performed by argon-induced fragmentation afterselection of the parent ion.

Eight peptides were predicted to derive from the product of the humantazarotene-induced gene (TIG)-2 (FIG. 22), covering 91 aminoacids out ofthe 143 amino acid-long sequence of the TIG-2 gene product (afterremoval of the predicted signal peptide). However, the C-terminalpeptide (peptide 8) was not tryptic lacking the last six amino acids ofthe predicted protein. This observation indicated that the activecompound might result from the proteolytic processing of the encodedprecursor (Table 1 and FIG. 22).

Table 1: Sequences of Peptides found in monoisotopic mass fingerprinting

The two peptides indicated with an asterisk were microsequenced by MS/MSfragmentation. The position of the peptides is defined in comparisonwith Preprochemerin amino acid sequence (SEQ ID NO: 8)

Residues # Sequence M + H  72-78 (K)LQQTSCR(K) 835.41 [SEQ. ID. NO: 15] 81-88 (R)DWKKPECK(V) 1033.51 [SEQ. ID. NO: 16]  29-39*(R)GLQVALEEFHK(H) 1270.68 [SEQ. ID. NO: 17]  98-109 (K)CLACIKLGSEDK(V)1279.64 [SEQ. ID. NO: 18] 114-125* (R)LVHCPIETQVLR(E) 1407.78 [SEQ. ID.NO: 19]  28-39 (R)RGLQVALEEFHK(H) 1426.78 [SEQ. ID. NO: 20] 126-137(R)EAEEHQETQCLR(V) 1472.64 [SEQ. ID. NO: 21] 141-157(R)AGEDPHSFYFPGQFAFS(K) 1904.02 [SEQ. ID. NO: 22]

Example 3 Cloning and recombinant expression of human Chemerin

In order to clone the Chemerin sequence (FIG. 6, GenBank Accession No.Q99969) a polymerase chain reaction (PCR) was performed on kidney cDNA(Clontech Laboratories). Primers were synthesized based upon the humanChemerin sequence and were as follows:

hChemerin fw: 5′ CAGGAATTCAGCATGCGACGGCTGCTGA 3′ SEQ ID NO: 23 hChemerinrv: 5′ GCTCTAGATTAGCTGCGGGGCAGGGCCTT 3′ SEQ ID NO: 24

Amplification was performed with Qiagen Taq polymerase in the conditionsdescribed by the supplier and with the following cycles: 3 min at 94°C., 35 cycles of 1 min at 94° C., 90 sec at 58° C. and 90 sec at 72° C.,followed by a final incubation of 10 min at 72° C. The amplificationresulted in a fragment of 500 bp containing the entire coding sequenceof the Chemerin gene. This fragment was subcloned into the vector pCDNA3(Invitrogen) for DNA sequencing analysis.

Maxiprep (Quiagen) DNA was used in transient transfections of HEK293cells expressing large T antigen (293T) and COS-7 cells using Fugene6 in10 cm plates. In parallel, transfections were performed in the same celllines with the expression vector alone (Mock transfected). 24 hoursafter transfection, the medium was replaced by 9 ml DMEM-F 12, 1% BSA,and 3 ml of supernatant were collected each 24 h for three days (48, 72and 96 h post transfection). CHO cells were transfected with the sameplasmid and transfected cells were selected with G418. The activity ofthe conditioned medium was verified on ChemerinR expressing cells usingthe aequorin assay.

Example 4 Recombinant Expression of Chemerin in Yeasts

The coding sequences of human and mouse Chemerin are amplified by PCRusing the following primers (Two different primers are used foramplification of 5′ end of human Chemerin to take into account thedifferent predictions of the signal peptide of this protein):

mChemerinf: 5′ TCTCTCGAGAAAAGAGAGGCTGAAGCTACACGTGGGACAGAGCCCGAA 3′ SEQID NO: 25 hChemerinaf:5′ TCTCTCGAGAAAAGAGAGGCTGAAGCTGGCGTCGCCGAGCTCACGGAA 3′ SEQ ID NO: 26hChemerinbf: 5′ TCTCTCGAGAAAAGAGAGGCTGAAGCTGTGGGCGTCGCCGAGCTCACG 3′ SEQID NO: 27 mChemerinr: 5′ AGGGAATTCTTATTTGGTTCTCAGGGCCCT 3′ SEQ ID NO: 28hChemerinr: 5′ AGGGAATTCTTAGCTGCGGGGCAGGGCCTT 3′ SEQ ID NO: 29

The amplified Chemerin sequences are cloned, sequenced and inserted inpPIC9K, a multicopy Pichia expression plasmid (InVitrogen) containingthe signals directing secretion of expressed proteins. Followingtransformation, Pichia pastoris cells are selected using G418antibiotic. After selection, 20 clones are analyzed for their expressionand the clone with the highest expression is amplified for large scaleexpression in shaker flasks. The medium is collected, centrifuged andused for partial purification with a protocol derived from the one usedfor Chemerin initial purification (see above).

Example 5 Recombinant Expression of Chimaeric Chemerin Fused withSecreted Alkaline Phosphatase (SEAP)

The coding sequences of mouse and human Chemerin are amplified by PCR,cloned and sequenced. PCR and sequencing primers are as follows:

mChemerinf: CAGGAATTCGCCATGAAGTGCTTGCTGA (SEQ ID NO: 30) hChemerinf:CAGGAATTCAGCATGCGACGGCTGCTGA (SEQ ID NO: 31) mChemerinr:GCTCTAGATTTGGTTCTCAGGGCCCTGGA (SEQ ID NO: 32) hChemerinr:GCTCTAGAGCTGCGGGGCAGGGCCTTGGA (SEQ ID NO: 33)

The cloned Chemerin sequences are then subcloned into the mammalianbicistronic expression vector, pCDNA3, to obtain a fusion protein withChemerin linked at its carboxy terminal end to secreted alkalinephosphatase, tagged with six histidine residues (His6). Mammalian cells,including COS-7, HEK-293 expressing the large T antigen (293 T) andCHO-K1 cells, are transfected with this plasmid using Fugene 6™ andincubated for 3-4 days in complete Ham's F12 medium (Nutrient MixtureHam's F12 (Life Technologies) containing 10% fetal bovine serum; 100IU/ml penicillin, 100 μg/ml streptomycin and 2.5 μg/ml fungizone(Amphotericin B). The supernatant containing Chemerin-SEAP-His6 iscollected after centrifugation, filtered (0.45 μm) and stored at 4° C.after adding 20 mM Hepes (pH 7.4) and 0.02% sodium azide.

For one-step affinity purification of the Chemerin fusion protein, thesupernatant is applied to 1 ml of Hisbond resin (Qiagen). After washing,bound Chemerin-SEAP-His6 is eluted with a gradient of imidazol. Theconcentration of isolated Chemerin-SEAP-His6 is determined by a sandwichtype enzyme-linked immunosorbent assay. Briefly microtiter plates arecoated with anti-placental alkaline phosphatase antibody. After blockingwith 1 mg/ml bovine serum albumin (BSA) in phosphate buffered saline,the samples are titrated and incubated for 1 h at room temperature.After washing, plates are incubated with biotinylated rabbitanti-placental alkaline phosphatase diluted 1:500 for 1 h at roomtemperature, washed again, and incubated with peroxidase-conjugatedstreptavidin for 30 min. After washing, bound peroxidase is reacted with3,3′,5,5′-tetramethylbenzidine. The reaction is stopped by adding 1NH₂SO₄, and absorbance at 450 nm is measured. Alkaline phosphataseactivity is determined by a chemiluminescent assay using the GreatEscape™ detection kit (Clontech). Purified placental alkaline phophataseis used to generate a standard curve. The enzymatic activity isexpressed as relative light units/sec.

Example 6 Quantitative RT-PCR

ChemeirnR and Chemerin transcripts were detected by quantitative RT-PCR(TaqMan) in total or polyA+ RNA samples from human tissues and bloodcell populations obtained commercially (Clontech and Ambion) or preparedlocally (RNeasy Mini Kit, Qiagen). Primers were 5′-GCAGACAAGCTGCCGGA-3′(SEQ ID NO: 34) as forward, 5′-AGTTTGATGCAGGCCAGGC-3′ (SEQ ID NO: 35) asreverse and 5′-AACCCGAGTGCAAAGTCAGGCCC-3′ (SEQ ID NO: 36) as probe forChemerin, 5′-GTCCCAGAACCACCGCAG-3′ (SEQ ID NO: 37) as forward,5′-AAGAAAGCCAGGACCCAGATG-3′ (SEQ ID NO: 38) as reverse and5′-TTCGCCTGGCTTACATGGCCTGC-3′ (SEQ ID NO: 39) as probe for ChemerinR and5′-GAAGGTGAAGGTCGGAGTC-3′ (SEQ ID NO: 40) as forward,5′-GAAGATGGTGATGGGATTTC-3′ (SEQ ID NO: 41) as reverse and5′-AGCTCTCCCGCCGGCCTCTG-3′ (SEQ ID NO: 42) as probe for the referencehousekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH).Standard curves were run systematically for the three genes, and thetranscript copy number of proChemerin and ChemerinR was normalized tothe GAPDH transcript copy number for each sample.

We investigated the presence of prochemerin and chemerinR transcripts invarious human tissues and leukocyte populations by real-time RT-PCR(Taqman). In addition to immature dendritic cells, chemerinR transcriptswere found primarily in spleen, lymph nodes and lung, and at lowerlevels in a number of other tissues (FIG. 24B). Abundant chemerintranscripts were found in liver, lung, pituitary and ovary (FIG. 24C),and lower levels could be detected in most tissues. Interestinglyhowever, no expression of chemerin was found in peripheral bloodleukocyte populations. Monoclonal antibodies generated against humanchemerinR by genetic immunization (as described in Costagliola et al.,1998), and characterized by FACS on CHO-K1 cell lines expressing thereceptor (data not shown) were used to confirm the presence of thereceptor at the surface of dendritic cells and macrophages. High levelsof chemerinR immunoreactivity were found on monocyte-derived immaturedendritic cells, and chemerinR was downmodulated following maturation ofthe cells as a result of LPS or CD40L stimulation (FIGS. 24, D and E).Similarly, chemerinR immunoreactivity was observed at the surface ofmonocyte-derived human macrophages (FIG. 24F).

Example 7 Functional Assay for ChemerinR

ChemerinR-expressing clones have been obtained by transfection of CHO-K1cells to coexpressing mitochondrial apoaequorin and Gα16, limitingdilution and selection by northern blotting. Positive clones were usedfor screening with human ovarian cancer ascites extracts prepared asdescribed above. A functional assay based on the luminescence ofmitochondrial aequorin intracellular Ca²⁺ release (Stables et al., 1997,Anal. Biochem. 252:115-126; incorporated herein by reference) wasperformed as described (Detheux et al., 2000, J. Exp. Med., 1921501-1508; incorporated herein by reference). Briefly, cells werecollected from plates in PBS containing 5 mM EDTA, pelleted andresuspended at 5×10⁶ cells/ml in DMEM-F12 medium. Cells were incubatedwith 5 μM Coelenterazine H (Molecular Probes) for 4 hours at roomtemperature. Cells were then washed in DMEM-F12 medium and resuspendedat a concentration of 0.5×10⁶ cells/ml. Cells were then mixed with testagonist peptides or plates containing tissue extracts and the lightemission was recorded for 30 sec using a Microlumat luminometer (PerkinElmer). Results are expressed as Relative Light Units (RLU).

FIG. 17 shows the concentration response curve for the truncatedPreprocHEMERIN peptide (SEQ ID NO: 73, FIG. 16) to ChemerinR expressedin CHO cells. The assay was carried out as described in the preceedingparagraph. As shown in the figure, the truncated PreprocHEMERIN moleculeactivates ChemerinR with an EC₅₀ of 4.27 nM. Results are expressed asRelative Light Units (RLU).

Example 8 Activation of Cells Expressing ChemerinR by RecombinantChemerin

The conditioned medium of COS-7, CHO-K1 and 293 T cells transfected withpCDNA3 encoding Chemerin or pCDNA3 alone, was collected and used foraequorin assays on CHO cells expressing ChemerinR. Results are shown inFIG. 14. Increasing amounts of conditioned supernatant resulted in anincrease in luminescence in aequorin system cells expressing ChemerinR.

Example 9 Production of Antibodies Specific for Chemerin and ChemerinR

Antibodies directed against Chemerin or ChemerinR were produced byrepeated injections of plasmids encoding Chemerin or ChemerinR intomice. Sera were collected starting after the second injection and thetitre and specificity of the antibodies was assessed by flowcytofluorometry with CHO-K1 cells transfected with the Chemerin orChemerinR cDNA and CHO-K1 cells transfected with the cDNA of unrelatedGPCR cDNA. Several sera were positive and were used forimmunohistochemistry and other related applications, including flowcytometry analysis of human primary cells.

Monoclonal antibodies were obtained from immune mice by standardhybridoma technology using the NSO murine myeloma cell line as immortalpartner. Supernatants were tested for anti ChemerinR antibody activityusing the test used for assessing the antisera. Cells from the positivewells were expanded and frozen and the supernatants collected.

In particular, BALB/c mice were injected with 100%1 g pcDNA-ChemerinR,or with the Chemerin C-terminal octapeptide FSKALPRS. Sera were testedby FACS on the CHO-ChemerinR cell line, or by ELISA for the Chemerinpeptide, and immune mice were used to generate monoclonal antibodies bystandard hybridoma technology, using the NSO myeloma cell line. The Igclass of selected hybridomas was determined with a mouse mAB isotypingkit (IsoStrip, Boehringer Mannheim).

FIG. 15 shows the results of experiments to characterize the antibodiesraised against ChemerinR. A mixture of recombinant cells made up of ⅔recombinant ChemerinR CHO cells and ⅓ mock-transfected CHO cells(negative control) was reacted with either a supernatant of cellsexpressing the anti ChemerinR 5C 1H2 monoclonal antibody (thick line) ora supernatant from cells with no known antibody activity (thin line,grey filling). After staining with FITC labeled anti mouse Ig thesepreparations were analyzed by flow cytofluorometry. Results aredisplayed as a histogram of the number of cells (Events axis) expressinga given fluorescence (FL1-H axis). Monoclonal 5C 1H2 allowed thediscrimination of the ChemerinR recombinant sub-population of cells fromthe negative control cells, as evidenced by the relative proportions ofboth types of cells. The background fluorescence of the assay is givenby the second staining (grey filling).

The ability of anti-chemerinR antibodies to block receptor activation bychemerin was investigated using the aequorin assay onchemerinR-expressing CHO-K1 cells. We found that two antibodies (4C7 and1H2) were able to efficiently inhibit calcium mobilization promoted byrecombinant chemerin, in a concentration-dependent manner (FIG. 26A).

Example 10 Binding Displacement Assay

For displacement experiments, ChemerinR-CHO-K1 cells (25,000 cells/tube)are incubated for 90 min. at 27° C. with 1 nM of SEAP-HIS6 orChemerin-SEAP-HIS6 in the presence of increasing concentrations ofunlabeled Chemerin in 250 μl of binding buffer (50 mM Hepes pH 7.4; 1 mMCa Cl₂; 0.5% Bovine Serum Albumin (BSA) Fatty Acid-Free; 5 mM MgCl₂).For saturation experiments, ChemerinR-CHO-K1 cells (25,000 cells/tube)are incubated for 90 min at 27° C. with increasing concentrations ofChemerin-SEAP-HIS6 in the presence or absence of 1 μM unlabeledChemerin. After incubation, cells are washed 5 times and lysed in 50 μlof 10 mM Tris-HCl (pH 8.0), 1% triton X100. Samples are heated at 65° C.for 10 min to inactivate cellular phosphatases. Lysates are collected bycentrifugation, and alkaline phosphatase activity in 25 μl of lysate isdetermined by the chemiluminescence assay described above.

Example 11 Competition Binding Assay

ChemerinR expressing CHO-K1 cells were collected from plates with PBSsupplemented with 5 mM EDTA, gently pelleted for 2 min at 1000×g, andresuspended in binding buffer (50 mM HEPES, pH 7.4, 1 mM CaCl₂, 5 mMMgCl₂, 0.5% BSA). Competition binding assays were performed in Minisorbtubes (Nunc), using the ¹²⁵I-YHSFFFPGQFAFS (SEQ ID NO: 91) peptide astracer (specific activity: 600 Ci/mmol, 50,000 cpm per tube), variableconcentrations of competitors, and 500,000 cells in a final volume of0.1 ml. Total binding was measured in the absence of competitor, andnonspecific binding was measured in the presence of a 100-fold excess ofunlabeled ligand. Samples were incubated for 90 min at 27° C. and thenbound tracer was separated by filtration through GF/B filters presoakedin 0.5% BSA. Filters were counted in a ®-scintillation counter. Bindingparameters were determined with the PRISM software (Graphpad Software)using nonlinear regression applied to a one-site competition model.

The structure-function analysis of peptides derived from the C-terminusof chemerin allowed to design a bioactive peptide (YHSFFFPGQFAFS (SEQ IDNO: 91), EC₅₀ of 28 nM on chemerinR-expressing CHO-K1 cells, using theaequorin-based assay) that could be labeled on its N-terminus tyrosinefor binding studies. This iodinated peptide was used in a competitionbinding assay, using the unlabeled peptide or recombinant chemerin ascompetitors. As shown in FIG. 23C, the Ki values were estimated to2.5±1.2 nM pKi: 8.82±0.38) for recombinant Chemerin (filled circles) and12.1±4.97 nM pKi: 7.95±0.18) for the unlabel peptide (open square) (mean±s.e.m for 3 independent experiments).

Example 12 Intracellular Cascade Assays

GTPγ³⁵S binding to membranes of cells expressing human ChemR23 wasperformed as described previously (Kotani et al. 2000). Briefly,membranes (10 μg) from CHO-hChemR23 cells, pretreated or not with PTX)were incubated for 15 min at room temperature in GTPγS binding buffer(20 mM HEPES pH 7.4, 100 mM NaCl, 3 mM MgCl₂, 3 μM GDP, 10 μg/mlsaponin) containing different concentrations of peptides in 96 wellmicroplates (Basic FlashPlates, New England Nuclear). [³⁵S]-GTPγS (0.1nM, Amersham-Pharmacia) was added, microplates were shaken for oneminute and further incubated at 300 for 30 min. The incubation wasstopped by centrifugation of the microplate for 10 min at 800 g and 40,and aspiration of the supernatant. Microplates were counted in aTopCount (Packard, Downers, Ill.) for 1 min per well. Functionalparameters were determined with the PRISM software (Graphpad Software)using nonlinear regression applied to a sigmoidal dose-response model.

The signaling pathways activated by chemerinR were investigated inCHO-K1 cells expressing the human receptor, but not G_(α16) orapoaequorin (CHO/chemerinR cells). Receptor activation was tested in aGTPγ[₃₅S] binding assay, using membranes from CHO/chemerinR cells andhuman chemerin. The results show stimulation of ChemerinR expressionCHO-K1 cells (EC₅₀: 7.8±0.4 nM, mean ±s.e.m for 4 independentexperiments, FIG. 23D). Furthermore, stimulation of these cells by humanChemerin at low nanomolar concentrations resulted in the release ofintracellular calcium and inhibition of cAMP accumulation (not shown),as well as phosphorylation of the p42 and p44 MAP kinases (FIGS. 23E andF). All these effects were inhibited by Pertussis toxin pretreatment,demonstrating the involvement of G_(i) family members. No activity ofrecombinant Chemerin or prochemerin was obtained in any of these assayson wild-type CHO-K1 cells (data not shown).

Example 13 Tissue Distribution of Chemerin and ChemerinR

Semi-quantitative RT-PCR was performed using gene-specific primers tohCHEMERIN and ChemerinR on polyA+ RNA and total RNA from various humantissues (CLONTECH and Ambion). Briefly, total RNA from blood cells wereprepared with Rneasy Mini Kit (Qiagen). The hCHEMERIN primers wereforward (5′-GCAGACAAGCTGCCGGA-3′; SEQ ID NO: 34), TaqMan probe(5′-AACCCGAGTGCAAAGTCAGGCCC-3′; SEQ ID NO: 36), and reverse(5′-AGTTTGATGCAGGCCAGGC-3′; SEQ ID NO: 35). The hChemerinR primers wereforward (5′-GTCCCAGAACCACCGCAG-3′; SEQ ID NO: 37), TaqMan probe(5′-TTCGCCTGGCTTACATGGCCTGC-3′; SEQ ID NO: 39), and reverse(5′-AAGAAAGCCAGGACCCAGATG-3′; SEQ ID NO: 38). Primers designed to thehousekeeping gene GAPDH Forward (5′-GAAGGTGAAGGTCGGAGTC-3′; SEQ ID NO:40), TaqMan pobe (5′-AGCTCTCCCGCCGGCCTCTG-3′; SEQ ID NO: 42), andreverse (5′-GAAGATGGTGATGGGATTTC-3′; SEQ ID NO: 41) were used toproduced reference mRNA profiles. The distribution of hCHEMERIN andChemerinR in various tissues is shown in FIGS. 18 and 19, respectively.The level of expression of hCHEMERIN or ChemerinR are expressed as aratio of hCHEMERIN or ChemerinR to GAPDH reference mRNA expression.

Example 14 Expression and Pharmacological Characterization of HumanChemerin

The recombinant Chemerin protein was purified by filtration through 0.45μm Millex filters (Millipore) and separation through a cation-exchangeHPLC column (Polycat 9.6×250 mm, Vydac, 0-1 M NaCl gradient in acetatebuffer pH 5). The protein concentration in active fractions wasdetermined following SDS/PAGE, by comparison with glutathioneS-transferase and lysozyme standards after silver staining.

Human Chemerin cDNA was cloned and expressed in CHO-K1 cells. Thebioactive recombinant protein was purified to homogeneity fromconditioned medium, and analyzed by mass spectrometry and SDS/PAGE,which confirmed C-terminal truncation after serine 157 (not shown). Amonoclonal antibody, generated against a peptide (FSKALPRS, SEQ ID NO:89) corresponding to the predicted C-terminal sequence of the geneproduct, was used to purify to homogeneity from CHO-K1 conditionedmedium, an unprocessed form of the protein (prochemerin), which wasconfirmed by mass spectrometry to retain the six C-terminal aminoacids(not shown). The amount of purified recombinant Chemerin (FIG. 23A) andprochemerin (not shown) was determined by comparison with proteinstandards, following SDS/PAGE and silver staining. It was inferred thatover 90% of prochemerin released by CHO-K1 cells was enzymaticallyprocessed into Chemerin. Comparison of the biological activity of thetwo purified proteins assayed in parallel on CHO-K1 cells expressinghuman ChemerinR (FIG. 23B) demonstrated that processed Chemerin (filledcircles) was about a hundred fold more active (EC₅₀: 4.5±0.7 nM, mean±s.e.m. for 7 independent experiments) than unprocessed prochemerin(open circles) (EC₅₀: 393±116 nM, mean ±s.e.m. for 3 independentexperiments). The N-terminus of prochemerin and Chemerin was determinedby mass spectrometry: a tryptic peptide (ELTEAQR, 845.45 Da, SEQ ID NO:90) corresponding to amino-acids 21 to 27 of preprochemerin wasidentified by sequencing (data not shown), confirming the signal peptidecleavage site predicted by the SignalP software from Expasy(http://www.cbs.dtu.dk/services/SignalP/). ChemerinR is structurally andevolutionary related to the C5a and C3a receptors, the prostaglandin D2receptor CRTH2, and the orphan GPR1 receptor (20). These receptors, aswell as a large set of other characterized and orphan receptors,including most chemokine receptors, were shown to be totally unreactiveto purified human Chemerin (data not shown). The activation of ChemerinRby a set of over 200 bioactive molecules, including all currentlyavailable chemokines, C5a, C3a, fMLP, bradykinin, PAF and leukotrienes,was also tested. All these agents were unable to promote receptoractivation even at concentrations significantly higher (100 nM or 1 μM)than those reported to activate their own receptors. Chemerin and itsreceptor appear therefore as a specific signaling system, in contrast tothe situation prevailing with inflammatory chemokines and theirrespective receptors. In order to investigate whether proteolyticactivation of prochemerin is performed intracellularly in the secretorypathway, or is an extracellular process, potentially regulated by theactivation of extracellular proteases, we tested the activation of humanpurified prochemerin in the medium of cultured cells and conditionedmedia. We could show that human prochemerin can be fully converted intoa form active on ChemerinR, during the incubation (at 100 nM) in theculture medium of hamster CHO-K1 cells, simian Cos-7 cells or humanHEK293 cells (data not shown), as well as in conditioned media fromthese cells (FIG. 24A). These data indicate that prochemerin processingis performed extracellularly, and that the active Chemerin product isnot degraded further by the proteolytic activity, and is thereforestable in extracellular medium. Although the protease responsible forthis processing is not known, the regulation of this enzyme activity isexpected to control the extracellular generation of active Chemerin invivo.

Example 15 High Affinity Activation of ChemerinR by C-Terminus TruncatedPeptide of Chemerin

In order to investigate the potential effect of peptides derived fromthe C-terminal domain of prochemerin, we first synthesized severalpeptides starting at position 139 of prochemerin, after the lastcysteine (Table 2), and tested their ability to trigger intracellularcalcium release in a cell line coexpressing the Chemerin receptor andapoaequorin, WE have use the arquorin assay as previously described inDetheux et al. (2000 J. Exp. Med. 192, 1501-1508). As shown in FIG. 25Aand Table 2, the peptide corresponding to the C-terminal end ofprochemerin (hProchemerin-25) was not able to activate the receptorunder high concentration (mean EC₅₀ of 160±21 μM), whereas the samepeptide lacking the 6 last amino-acids (hChemerin-19) activated theChemerin receptor with very high affinity (mean EC₅₀ of 16.7±3.2 nM). Asdescribed before, the recombinant prochemerin was poorly active (meanEC₅₀ of 393±116 nM) compared to the affinity of the processedrecombinant Chemerin (mean EC₅₀ of 4.5±0.7 nM). These results areconsistent with the previously data showing the functional importance ofthe C-terminal processing of the prochemerin, which allows thetransformation of a low affinity precursor to a high affinity form ofthe ligand. Surprisingly, these data also suggest that a sequencecorresponding to the last 19 C-terminus amino acids of Chemerin seems tobe sufficient for providing high affinity receptor activation.

To study the accuracy of the processing of the immature form ofChemerin, we further investigated the effect of C-terminal truncatedpeptides variants (Table 2). As shown in FIG. 25B and Table 2, additionof a single amino-acid (h[Lys-20] Chemerin-19) to the C-terminal end ofthe control peptide strongly affected the affinity (EC₅₀ of 170 μMcompared to a value of 16.7±3.2 nM for hChemerin-19). The same effectwas observed after removal of at least 2 amino-acids (h[Phe18Ser19]Chemerin-19, EC₅₀ of 220 μM; h[Ala17Phe18Ser19] Chemerin-19, EC₅₀ of130±10 μM). However, removal of only one amino-acid slightly impairedthe response (h[Ser19] Chemerin-19, EC₅₀=97±13 nM). From these data, theC-terminal end of the Chemerin appeared to be extremely precise, asaddition of only one amino acid abrogated the high affinityintracellular calcium response. We also showed the functional importanceof the Phenylalanine residue in position 18 and, more slightly, theSerine in position 19. Thus, C-terminal modification of the Chemerinseriously impaired the high affinity activation of its receptor,demonstrating the accuracy of the activating cleavage.

TABLE 2 The EC₅₀ value of the truncated Chemerin peptide SEQ ID NOPeptide (name and sequence) Mean EC50 52 Human prochemerin-25   160 ± 21μM QRAGEDPHSFYFPGQFAFSKALPRS 53 Human Chemerin-19  16.7 ± 3.2 nMQRAGEDPHSFYFPGQFAFS 54 Human [Lys20] Chemerin-19 170 μMQRAGEDPHSFYFPGQFAFSK 55 Human [ΔSer19] Chemerin-19    97 ± 13 nMQRAGEDPHSFYFPGQFAF 56 Human [ΔPhe18Ser19] Chemerin-19 220 μMQRAGEDPHSFYFPGQFA 57 Human [ΔAla17Phe18Ser19]   130 ± 10 μM Chemerin-19QRAGEDPHSFYFPGQF 58 Human [ΔPhe16Ala17Phe18Ser19] inactif Chemerin-19QRAGEDPHSFYFPGQ 59 Human Chemerin-7   220 ± 100 μM PGQFAFS 60 HumanChemerin-8     2 ± 1 μM FPGQFAFS 61 Human Chemerin-9     7 ± 0.25 nMYFPGQFAFS 62 Human Chemerin-10   8.2 ± 2 nM FYFPGQFAFS 63 HumanChemerin-12  12.2 ± 3.4 nM HSFYFPGQFAFS 64 Human Chemerin-13 14 nMPHSFYFPGQFAFS 65 Human [Ala-1] Chemerin-9   496 ± 80 nM AFPGQFAFS 66Human [Ala-2] Chemerin-9 155.3 ± 41.6 nM YAPGQFAFS 67 Human[Ala-3] Chemerin-9  42.5 ± 7.5 nM YFAGQFAFS 68 Human [Ala-5] Chemerin-9 35.8 ± 5.9 nM YFPGAFAFS 69 Human [Ala-6] Chemerin-9     5 ± 1 μMYFPGQAAFS 70 Human [Ala-8] Chemerin-9    38 ± 7 μM YFPGQFAAS 71 Human[Ala-9] Chemerin-9  48.3 ± 5.7 nM YFPGQFAFA

Example 16 The Shorter C-Terminal Nonaoeptide YFPGOFAFS has a HighAffinity on ChemerinR

We then determined the minimum length of the C-terminal fragment able toactivate the Chemerin receptor with high potency. Successive truncationsof the N-terminal domain of the hChemerin-19 peptide were synthesizedand tested using the aequorin assay (FIG. 25C). Truncations from residue1 to residue 10 (hChemerin-17 to hChemerin-9, FIG. 20 and EC₅₀ values intable 2) did not affect intracellular calcium signaling. However,removal of the Tyrosine residue in position 11 (hChemerin-8) resulted ina severely loss of affinity for the receptor (EC₅₀ of 2±1 μM compared toa value of 16.7±3.2 nM for the control peptide: Human chemerin-19), andthe response was completely abrogated for shorter peptide (hChemerin-7,EC₅₀ of 220±100 μM). These results indicated that only the last 9 aminoacids of Chemerin are necessary for high affinity receptor activation,as the EC₅₀ of the nonapeptide is 7±0.25 nM, which is in the same rangeto the affinity of the recombinant Chemerin.

Example 17 Aromatic Residues in Chemerin C-Terminus are Necessary forChemerinR Activiation

Since multiple residues within the last 9 amino acids sequence ofChemerin appeared to be important for receptor activation, we examinedthe relative contribution of each amino acid of the YFPGQFAFS peptide inChemerin receptor activation, by using an alanine-scanning mutagenesisapproach. Eight different alanine-substituted hChemerin-9 analogs weresynthesized and tested for intracellular calcium accumulation. As shownin FIG. 25D and Table 2, the EC₅₀ of the Q5A, P3A and S9A mutatedpeptides was shifted to higher concentrations (EC₅₀ of 35.8±5.9 nM,42.5±7.5 nM and 48.3±5.7 nM respectively) as compared with the controlpeptide (mean EC₅₀ of 7±0.25 nM). The EC₅₀ of the F2A and Y1A peptideswas more severely affected (EC₅₀ of 155.3±41.6 nM and 496±80 mM,respectively), and alanine substitution of Phe 6 and Phe 8 dramaticallyimpaired the functional response of Chemerin receptor (EC₅₀ of 5±1 μMand 38±7 μM, respectively). These data suggested that aromatic Y1, F2,F6 and F8 residues play an important role in receptor activation.

Example 18 Chemotaxis and Ca²⁺ Mobilization Assays on Primary Cells

Monocyte-derived DCs were generated by GM-CSF (50 ng/ml) and IL-13 (20ng/ml) stimulation as previously described (17). Maturation of DCs wasachieved following stimulation with 100 ng/ml LPS. Macrophages wereobtained by incubating monocytes in Petriperm dishes (Haereus) for 6days in RPMI supplemented with 10% FCS and 10 ng/ml MCSF. Cell migrationwas evaluated using a 48-well microchemotaxis chamber technique asdescribed (18). For Ca₂₊ mobilization assays, monocyte-derived DCs ormacrophages (10⁷ cells/ml in HBSS without phenol red but containing 0.1%BSA) were loaded with 5 μM FURA-2 (Molecular Probes) for 30 min at 37°C. in the dark. The loaded cells were washed twice, resuspended at 10⁶cells/ml, kept for 30 min at 4° C. in the dark with or without theblocking 4C7 monoclonal antibody (10 μg/ml), and transferred into thequartz cuvette of a luminescence spectrometer LS50B (PerkinElmer). Ca²⁺mobilization in response to recombinant Chemerin was measured byrecording the ratio of fluorescence emitted at 510 nm after sequentialexcitation at 340 and 380 nm.

The biological function of Chemerin was further investigated onleukocyte populations. In accordance to the coupling of human ChemerinRthrough the G_(i) class of G proteins, its structural relatedness tochemoattractant receptors, and its expression in antigen-presentingcells, we showed that Chemerin acted as a chemotactic factor for thesecells. Dendritic cells and macrophages were differentiated in vitro fromhuman monocytes. Human recombinant Chemerin promoted in vitro migrationof macrophages and immature dendritic cells (FIGS. 26 B, C, and F),whereas no chemotaxis of mature dendritic cells was observed (data notshown). Maximal chemotactic responses were obtained for concentrationsof 100 pM to 1 nM, according to the batch of recombinant Chemerin. Suchbell-shaped chemotactic response, with a maximum corresponding toconcentrations below the EC₅₀ observed in other functional assays, istypically observed for other chemotactic factors such as chemokines. Theeffect was completely abolished following treatment with Pertussis toxin(FIGS. 26C and F), demonstrating the involvement of the G_(i) class of Gproteins. Migration of macrophages and dendritic cells was alsoinhibited by the antiChemerinR monoclonal antibody 4C7 (FIGS. 26C and F)without affecting RANTESinduced cell migration, demonstrating that theeffect is specifically mediated by the ChemerinR. A checkerboardanalysis showed that, when equal concentrations of Chemerin were presentin both the lower and upper wells, no significant increase in cellmigration was observed (FIGS. 26C and F). Thus, the migration ofmacrophages and immature dendritic cells induced by Chemerin isessentially a chemotactic effect rather than chemokinesis. We alsoinvestigated whether recombinant Chemerin could induce Ca²⁺ mobilizationin antigen-presenting cells. As expected, intracellular Ca²⁺ levelsincreased in immature dendritic cells in response to recombinantChemerin (FIG. 26D), whereas the 4C7 antibody inhibited the Ca²⁺response (FIG. 26E). Similar observations were made for macrophages(FIGS. 26G and H).

Example 19 Bioactive Chemerin Concentration in Human Samples

In order to investigate whether chemerin is frequently generated inpathological situations in human, we fractionated a set of inflammatoryfluids and assayed the chemerin content by measuring the biologicalactivity of the fractions on chemerinR, as compared to a standard curvemade with purified recombinant chemerin. Significant levels of activechemerin, well within the active range (33 to 358 ng/ml, correspondingto 2 to 23 nM), were found in the majority of ascitic fluids resultingfrom ovary cancer, but also in ascitic fluids resulting from a livercancer and from an ovary hyperstimulation syndrome, as well as in a poolof articular fluids from arthritic patients (Table 3). Interestingly,active chemerin was not detected in articular fluid pooled from patientswith arthrosis (Table 3), nor in fractions from human hemofiltrate (notshown), demonstrating that its presence is linked to inflammatorysituations.

The amount of Chemerin in ascitic (samples 1-17) and articular (samples18 and 19) fluids was estimated following two fractionation steps, byassaying the fractions on ChemerinR-expressing cells, using theaequorin-based assay and a standard curve made with purified recombinanthuman Chemerin. Articular fluids from arthritis and arthrosis patientswere pooled for measurement, following centrifugation. 0. H .S.: ovarianhyperstimulation syndrome. n.d.: not detectable (the limit of detectionin the assay conditions is given).

TABLE 3 Bioactive Chemerin concentration in human samples. ChemerinSample Pathology (ng/ml) 1 Ovary Carcinoma 74 2 Ovary Carcinoma 73 3Ovary Carcinoma 104 4 Ovary Carcinoma 92 5 Ovary Carcinoma n.d. (<10) 6Ovary Carcinoma 82 7 Ovary Carcinoma 103 8 Ovary Carcinoma 43 9 OvaryCarcinoma 87 10 Ovary Carcinoma n.d. (<10) 11 Ovary Carcinoma 90 12Ovary Carcinoma 33 13 Ovary Carcinoma 57 14 Ovary Carcinoma 87 15 OvaryCarcinoma 62 16 Ovary Carcinoma 37 17 O.H.S. 116 18 Arthritis 358 19Arthrosis n.d. (<1) 

Example 20 In Vivo Gene Therapy in Mouse

B16-F0 Melanoma Model.

B 16-F0 melanoma cells (ATCC) were transfected with the pEFIN3-mousechemerin plasmid using FuGene6, and selected with 800 μg/ml G418. Cloneswere characterized by assaying the conditioned medium onchemerinRexpressing cells. In vitro proliferation rate was determined byBrdU incorporation as described₃₀. For in vivo studies, cells werewashed twice with PBS, and grafted (6×10₅ cells in 0.1 ml PBS)subcutaneously into the back of 10-week-old C57B16 mice (5 to 11 miceper group, Harlan, The Netherlands). Perpendicular tumor diameters (Dand d) were measured every 2 days, and the volume was estimated asV=π(d/2)(D/2)(d/2). Statistical analysis was performed by using theunpaired non parametric Mann-Whitney test. For microscopic analysis,tumors were embedded in OCT, snap-frozen in −80° C. isopentane and cutat 12 μm. Sections were stained with hematoxylin-eosin (HE) for routineanalysis. All animal procedures were approved by the ethical committeeof the Medical School of the Université Libre de Bruxelles.

The biological function of Chemerin was further investigated in a mousemodel in vivo. In accordance to the coupling of human ChemerinR throughthe G₁ class of G proteins, its structural relatedness tochemoattractant receptors, and its expression in dendritic cells,Chemerin acted as a chemotactic factor for these cells. Dendritic cellswere differentiated in vitro from human monocytes. Human recombinantChemerin was chemotactic in vitro for immature, but not mature,dendritic cells, with a maximal activity at 1 nM (See example 18 of thepresent application).

As active Chemerin was originally isolated from tumoral ascitis, weevaluated the significance of this expression in a tumor context, byinvestigating the consequence of Chemerin expression in a mouse tumormodel in vivo. The mouse prochemerin and ChemerinR cDNAs were cloned.Following their expression in CHO-K1 cells, functional assaysdemonstrated that the human and mouse recombinant ligands were equallyactive on both the human and mouse receptors (data not shown). Themelanoma cell line B16F0 was transfected with a bicistronic expressionvector containing the mouse Chemerin cDNA (or a control vector), andstable cell lines were established. The expression of bioactive Chemerinwas confirmed by measuring the activity of conditioned medium. The twoselected cell lines released over a period of 24 hours about 125 ng/mlactive Chemerin in the culture medium. Expression of Chemerin did notmodify the growth rate of the cell lines, as assessed by measuring theproportion of cells in the various phases of the cell cycle (FIGS.27A-C), or by directly counting cells over time (data not shown).However, following subcutaneous graft of the cells to syngenic mice, thephenotype of the developing tumors was profoundly modified by Chemerinexpression. In three independent series, all mice receiving wild-type B16F0 cells developed a rapidly growing tumor, in accordance with theliterature, while a number of mice receiving Chemerin-expressing cellsdid not develop tumors up to four weeks after the graft. By combiningthe three series, 5 out of 24 mice grafted with Chemerin-expressingcells did not develop tumors (versus 0/24 in the control group, p<0.05,Fisher test). The size of the developing tumors was also much smallerfor the Chemerin group (an average reduction of 70% 21-24 days after thegraft of cells, FIG. 27D). The difference was significant from day 10after the graft (p=0.02 to 0.004 according to time points,non-parametric Mann-Whitney test). Macroscopic analysis at the end ofthe observation period (12 to 30 days) revealed a number of phenotypicdifferences between the two groups. Chemerin-producing tumors werecharacterized by a more abundant vascularization, and a much lowerextent of necrotic areas. These phenotypic differences were not theconsequence of a difference in the size of the tumors, as they wereobserved as well following the selection of rare size-matched tumorsbelonging to the two groups. Microscopic analysis, followinghematoxylin-eosin staining, confirmed these observations, particularlythe major difference in the extent of necrosis, that occupies thelargest part of control tumors, while being rare in Chemerin-producingtumors (FIGS. 27E and F).

Example 21 Calcium Flux—The Aequoscreen™ Assay

ChemerinR expressing clones were transfected to coexpress mithochondrialapoaequorin and Gα16. Cells in mid-log phase, grown in media withoutantibiotics 18 hours prior to the test, were detached by gentle flushingwith PBS-EDTA (5 mM EDTA), recovered by centrifugation and resuspendedin “BSA medium” (DMEM/HAM's F12 with HEPES, without phenol red +0.1%BSA). Cells were then counted, centrifuged and resuspended in a 15 mlFalcon tube at a concentration of 1×10⁶ cells/ml.

Coelenterazine h (Molecular Probes, cat No. C-6780, stock solution: 500μM in Methanol) was added to the cells in suspension at a finalconcentration of 5 μM.

The Falcon tube, wrapped in aluminium foil, was then placed on avertical rotating wheel and incubated at room temperature (temperatureshould be maintained below 22° C.) overnight in order to reconstituteactive aequorin.

Cells were then diluted 1/10 in “BSA-medium” and incubated as describedabove for 60 min. Reference ligands were diluted in “BSA-medium” anddistributed in a 96-well plate (50 μl/well). For each measurement, 50 μlof cells (i.e. 5 000 cells) were injected into each well of the platecontaining the ligands, and the emitted light is recorded (FDSS,Hamamatsu) during 20 seconds following cells injection. Results wereexpressed as Relative Light Units (RLU). Digitonin (50 μM, Sigma, catn^(o)37006) is used as positive controls of the cell response.

The intensity of the emitted light was integrated, yielding for eachwell one value representative of the emitted light.

Example 22 Aromatic and Hydrophobic Residues in N-Terminus of theNonapeptide YFPGQFAFS are Necessary for ChemerinR Activation

In order to investigate the importance of the N-terminus part of thenonapetpide YFPGQFAFS, several peptides were synthesized and testedusing the Calcium flux-Aequoscreen assay. As shown in FIGS. 28 to 32 andTable 4, peptides containing an aromatic or hydrophobic residue onposition N1 and N2 are in the same range of activity of the recombinantChemerin. These data suggested that aromatic and hydrophobic residuesplay an important role in receptor activation.

TABLE 4 EC5O value of the peptides modified by hydro- phobic residue(N-terminus) and EC50 value of the peptide modified in position 9 by anAspartate residue and an amide function. SEQ ID NO. Sequence EC50 (nM)92 LFPGQFAFS 66.3 93 IFPGQFAFS 25.8 94 FLPGQFAFS 29.1 95 YLPGQFAFS 3.2396 YVPGQFAFS 43.8 97 YFPGQFAFD-CONH2 4.3

Example 23 Modification of the C Terminal Part of the NonapeptideYFPGQFAFS

In order to clarify the role of the C-terminal part in the interactionwith ChemerinR, the Serine on position 9 was mutated by an Aspartateresidue and the carboxylic group on the C-term was replaced by an amidefunction. This peptide was tested using the Calcium flux-Aequoscreenassay. As shown in FIG. 33 and Table 4, this modification led to theidentification of a peptide having an activity in the same range of therecombinant Chemerin.

Other Embodiments

The foregoing examples demonstrate experiments performed andcontemplated by the present inventors in making and carrying out theinvention. It is believed that these examples include a disclosure oftechniques which serve to both apprise the art of the practice of theinvention and to demonstrate its usefulness. It will be appreciated bythose of skill in the art that the techniques and embodiments disclosedherein are preferred embodiments only that in general numerousequivalent methods and techniques may be employed to achieve the sameresult.

All of the references identified hereinabove, are hereby expresslyincorporated herein by reference to the extent that they describe, setforth, provide a basis for or enable compositions and/or methods whichmay be important to the practice of one or more embodiments of thepresent inventions.

1. A polypeptide comprising an amino acid sequence selected from thegroup consisting of LFPGQFAFS, IFPGQFAFS, FLPGQFAFS, YLPGQFAFS,YVPGQFAFS and YFPGQFAFD-CONH2, wherein the polypeptide bindsspecifically to a ChemerinR polypeptide.
 2. A nucleic acid sequenceencoding the amino acid sequence of claim
 1. 3. The nucleic acid ofclaim 2, said nucleic acid contained in an expression vector.
 4. Thenucleic acid of claim 3, wherein the expressing vector is a plasmid DNAexpression vector.
 5. The nucleic acid of claim 3, wherein theexpressing vector is an adenovirus vector comprising the coding sequenceunder the control of tissue specific, tumor selective promoter.
 6. Acomposition comprising the polypeptide of claim
 1. 7. A method ofinhibiting cell proliferation comprising administering to a cell thecomposition of claim
 6. 8. A method of inhibiting cell proliferationcomprising administering to a cell a composition comprising apolypeptide comprising the sequence of SEQ ID NO: 14, or SEQ ID NO: 61.9. A method of inhibiting cell proliferation comprising administering toa cell the expression vector of claim 3